- Email: [email protected]
The biology of steroid hormones and endocrine treatment of breast cancer
ARTICLE IN PRESS The Breast (2005) 14, 452–457
THE
BREAST www.elsevier.com/locate/breast
ORIGINAL ARTICLE
The biology of steroid hormones and endocrine treatment of breast cancer Mitch Dowsett, Elizabeth Folkerd, Deborah Doody, Ben Haynes Department of Biochemistry, Royal Marsden Hospital, London SW3 6JJ, UK
KEYWORDS Oestrogen; Breast cancer; Aromatase
Summary Recognition of the role of oestrogens in the stimulation of breast tumour growth has led to the development of several therapies based on endocrine intervention. Endocrine agents are currently used as a treatment for steroid receptor-positive breast cancer and more recently as a preventative measure in high-risk populations. Accurate quantification of resulting steroid hormonal profiles is essential to the understanding of the biological action and efficacy of these agents. In premenopausal women GnRH agonists suppress ovarian oestrogen synthesis and reduce oestradiol to close to postmenopausal levels. GnRH agonists used in combination with an aromatase inhibitor suppress levels even further. In contrast, tamoxifen can lead to markedly enhanced levels. In postmenopausal women aromatase inhibitors can achieve an almost complete inhibition of the aromatase enzyme. & 2005 Elsevier Ltd. All rights reserved.
Introduction The stimulation of breast cancer growth by oestrogens has provided a long established target for several well-tolerated treatments for steroid receptor-positive breast cancer and in recent years this has extended to their exploratory evaluation in the prevention context. In some cases the therapies used are specifically aimed at reducing oestrogen synthesis while in other cases the aim is to antagonise oestrogen action, but this can have secondary consequences for steroid hormone proCorresponding author. Tel.: +44 20 7808 2887;
fax: +44 20 7376 3918. E-mail address: [email protected] (M. Dowsett).
files as a result of feed-back mechanisms. An understanding of these effects is important for an appreciation of the mechanism of action of our most important endocrine agents and to allow their rational application in different clinical contexts.
Oestrogen synthesis In premenopausal women the large majority of plasma oestrogen synthesis is in the ovaries under the control of stimulatory effects of LH and FSH. Levels vary greatly during the menstrual cycle, ranging from 100 pmol/l or less in the very early follicular phase to values over 1000 pmol/l shortly
0960-9776/$ - see front matter & 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.breast.2005.08.019
ARTICLE IN PRESS The biology of steroid hormones and endocrine treatment of breast cancer prior to ovulation. Overall mean levels approximate to between 300 and 400 pmol/l. A small proportion of plasma oestrogens are derived from the peripheral synthesis by aromatase that is present in numerous non-glandular tissues, in particular subcutaneous fat. On average these peripheral sources make up less than 10% of the oestrogen in the circulation of premenopausal women, although it may be substantially greater in some obese women. Synthesis also occurs in the normal breast and in breast carcinomas and this source may be of particular relevance to the oestrogens that reach malignant breast cells. In postmenopausal women the ovaries cease their production of oestrogens as a result of the complete loss of primordial follicles. Oestrogens, however, continue to be synthesised by peripheral aromatisation from androgens that reach the circulation from both the adrenals and the ovaries. Peripheral aromatase activity increases with age such that this source of oestrogen approximately doubles in comparison to the same source in premenopausal women.1 Using the most sensitive assays (see later) plasma oestradiol levels approximate to mean levels of about 20 pmol/l in postmenopausal women.2,3 Oestradiol is the most potent of the nonconjugated oestrogens. It enters cells by free diffusion although its intracellular concentration is enhanced in those organs and tissues that express oestrogen receptors (ERs). The passage into the cells is impeded by the binding of oestradiol to albumin at low affinity and to sex hormone binding globulin (SHBG) at high affinity. Oestradiol is converted reversibly to oestrone, and oestrone itself is converted reversibly to oestrone sulphate. This latter conjugated oestrogen is not able to bind directly to the oestrogen receptor, but it may form an important reservoir for conversion to the primary oestrogens by the action of estrone sulphatase (Fig. 1).
androstenedione
testosterone
Steroids and the risk of breast cancer During the 1970s and 1980s many case-control studies were conducted to determine whether plasma steroid levels were related to a greater risk of developing breast cancer. However, the difficulty of avoiding bias in these studies resulted in a confused literature. More recently, a number of prospective studies have reported more consistent data and have culminated in an overview of nine such studies.4 This analysis revealed highly significant relationships between breast cancer risk and plasma oestradiol, oestrone, testosterone and androstenedione levels in postmenopausal women. A modest negative but significant relationship was found with SHBG. The relationship with oestradiol has been shown to largely explain the risk found between body mass index and breast cancer risk in postmenopausal women.5 These findings have led to the consideration that measurement of such hormone levels in postmenopausal women may enable them to be fitted into an algorithm of breast cancer risk.6 We have considered the possibility that plasma hormone levels might be subject to genetic controls and vary according to genetic polymorphisms in enzymes controlling their synthesis and metabolism.3 If so, this would provide indirect evidence for the involvement of these genes in breast cancer. We found that there was a highly significant relationship between plasma oestradiol and an even stronger relationship for plasma oestradiol/ testosterone ratio with the presence of a polymorphism in exon 10 of CYP19 (aromatase). We also established that there was a significant relationship between the plasma levels of SHBG and a polymorphism in the 50 -untranslated region of the SHBG gene. While these studies revealed highly statistically significant findings the proportion of the variability of the plasma hormone levels explained by the polymorphisms was small. At present it is unlikely that the assessment of these polymorphisms on their own would be useful in the evaluation of breast cancer risk and indeed they have not been shown to have a direct relationship with risk in relatively large-scale studies.3
SHBG
AROMATASE oestrone
453
oestradiol
oestrone sulphate
Figure 1 Steroids with major involvement in breast cancer. Testosterone and oestradiol bind with high affinity to sex hormone binding globulin (SHBG).
Premenopausal treatments Ovariectomy leads to a steroid hormone milieu that is very similar to that in postmenopausal women although oestrogen values may be marginally below those in postmenopausal women since the ovarian source of androgens is ablated by this operation. GnRH agonists are now a very popular means of
ARTICLE IN PRESS 454
M. Dowsett et al.
achieving a so-called medical ovariectomy. It can be seen in Fig. 2 that after 1 month’s treatment with 3.6 mg/month of goserelin in 20 patients with endometriosis, plasma oestradiol levels were reduced in all patients to mean values of 18 pmol/l.7 Thereafter, measurements made just prior to each of the monthly injections revealed that in virtually all cases the oestradiol levels remained below 100 pmol/l, but in many patients there was a minor degree of recovery above the 1-month levels, probably due to a low level of follicular oestrogenesis. In this series there was also one patient who showed a complete recovery of premenopausal levels. This particular patient showed a reaction around the injection site. Further studies revealed that this is a very uncommon but not unique example of such an absence of maintained suppression. When tamoxifen is administered to premenopausal women very high levels of oestradiol (sometimes as high as 3000 pmol/l or more) can occur.8 It is clear that despite this, tamoxifen remains an effective agent in premenopausal women, with similar activity to that achieved by ovariectomy or GnRH agonists. Calculations can be made on the competition between oestradiol with tamoxifen and its metabolites at the ER.9 These indicate that even in the presence of 1500 pmol/l of oestradiol 600
Plasma oestradiol (pmol/l)
500
400
300
200
100
0 Pre
1
2
3 4 Time (months)
5
6
Figure 2 Effect of goserelin (3.6 mg/month) on plasma oestradiol levels in 20 premenopausal endometriosis patients. Data summarised in Dowsett et al.7
(taken as a mean level in tamoxifen-treated premenopausal women) there is between 98% and 99% occupancy of the ER by tamoxifen such that the effective concentration of oestradiol on the receptor would be about 25 pmol/l. This would be consistent with a 10- to 20-fold reduction in oestradiol stimulation from baseline in such patients. When GnRH agonists and tamoxifen are given in combination there is no such increase in plasma oestradiol levels; rather the levels are similar to those seen with GnRH agonists alone and may be marginally less.8 In these circumstances tamoxifen would be expected to exert a full competition with oestradiol and therefore the combination could be expected to be more effective than either agent alone. This has been demonstrated to be the case in the small number of clinical trials conducted to date.8 Aromatase inhibitors are contra-indicated in premenopausal patients largely because of the expectation that they might cause cystic ovaries and lead to incomplete aromatase inhibition. This is rational and there is preclinical evidence of it, but the clinical experience to demonstrate this is in fact slight. We have reported a study of the triazole aromatase inhibitor YM511 in 30 premenopausal primary breast cancer patients who took part in a multi-centre, placebo-controlled, double-blind, randomised study in the 2 weeks prior to surgery.9 It was clear in this study that the expected increase in luteinising hormone (LH) and follicle-stimulating hormone (FSH) levels occurred in most patients and nearly all patients showed increases in testosterone and androstenedione as a result of this increased stimulation and aromatase inhibition. Effects on oestradiol levels were very variable with five of the on-treatment values in the 19 patients on YM511 being above the normal range of 100–900 pmol/l and five being below (Fig. 3). For oestrone, which is more dependent on peripheral synthesis, there were a greater number of patients with suppressed levels (Fig. 3). No suppression was found on the expression of the proliferation-associated nuclear antigen Ki67 in the tumours of these patients. This is in stark contrast to the suppression of Ki67 in the breast carcinomas of all but four of the 56 ERpositive, anastrozole-treated, postmenopausal patients over a similar 2-week period. These data therefore enhance the view that aromatase inhibitors are likely to have modest if any anti-tumour efficacy in premenopausal women. The use of aromatase inhibitors in combination with GnRH agonists is an area of increasing interest. We showed that the second generation aromatase inhibitor formestane suppressed the plasma levels
ARTICLE IN PRESS The biology of steroid hormones and endocrine treatment of breast cancer E1 (pmol/l)
E2 (pmol/l) 1500
600
1000
400
500
200
0
Nil
YM511 2w
0
Nil
YM511 2w
Figure 3 Comparison of plasma oestradiol and oestrone levels in premenopausal women after no treatment or 2 weeks’ treatment with YM511.9
of oestradiol consistently below 10 pmol/l in five premenopausal patients who were also treated with goserelin.10 We obtained similar data when determining the effect of the triazole aromatase inhibitor vorozole in combination with goserelin.11 Thus the combination of a GnRH agonist and an aromatase inhibitor appears to be a viable approach to obtaining near complete oestrogen deprivation in premenopausal women.
Postmenopausal treatments In postmenopausal women the endocrine effects of tamoxifen are much more modest than those in premenopausal women. There are increases in sex hormone binding globulin that would be expected to enhance the binding of plasma oestradiol and reduce its biological activity. The relationship between SHBG concentrations and residual free oestradiol is, however, relatively flat such that these changes, although to the advantage of the overall suppression of oestrogen stimulation of breast cancer, are probably only a minor contributor. There are increases in oestrone sulphate levels by about 20% and small decreases in plasma oestradiol levels (by about 10%), the latter probably being secondary to a fall in plasma testosterone due to a reduced ovarian secretion of this androgen.12 All of these changes in plasma steroids are probably of little importance in postmenopausal women treated with tamoxifen since it can be estimated that there is greater than 99.9% occupancy of ER by tamoxifen and its metabolites in this population.9 The third generation aromatase inhibitors achieve near complete inhibition of the enzyme in postmenopausal women. A cross-over study with 12
455
patients treated with both anastrozole and letrozole revealed that aromatase activity was suppressed to below 1% of baseline activity in all 12 patients by letrozole, but in only one patient on anastrozole in whom the mean residual levels were 2.7%.2 These latter levels were thus similar to those with exemestane at its clinical dose of 25 mg/day.13 In the same cross-over study of anastrozole/ letrozole all of the oestradiol levels were suppressed to below the detection limit of the assay (2.1 pmol/l) with letrozole, but not with anastrozole.2 However, the difference was not statistically significant. For oestrone and oestrone sulphate significant differences were seen with letrozole showing a greater suppression in both cases. Consideration of the clinical data between these two agents, however, suggests that these differences in pharmacological effectiveness are probably of only modest, if any, clinical significance. It is unusual when using highly sensitive oestradiol assays to find patients that do not show suppression to below or close to the detection of these assays. In one study14 we observed 2 out of 29 patients who did not show suppression at all after 3 months of letrozole treatment. In one of these patients drug levels were found to be low and compliance may have been poor. However, normal drug levels were seen in the other patient and an alternative explanation needs to be sought. It should be stressed, however, that in our experience less than 1% of postmenopausal patients do not show good suppression with modern inhibitors.
Problems of plasma oestradiol measurement in postmenopausal women We have recently published data illustrating the very poor performance of some plasma oestradiol assays that are in widespread routine use for the measurement of postmenopausal levels.15 In the main, these assays have been developed for the assessment of premenopausal levels and in association with ovulation induction schedules where very high levels are observed. Figure 4 summarises our observations and it is clear that the plasma levels seen with two such assays in which the oestrogen is not extracted from the plasma showed mean levels of about 100 pmol/l and very modest suppression with the aromatase inhibitor. This contrasts markedly with the mean levels seen when using a methodology that has been optimised for postmenopausal measurements. The major bias in the nonextraction assays is particularly clear when comparison is made with results obtained when oestradiol is extracted from the serum and assayed
ARTICLE IN PRESS 456
M. Dowsett et al.
Figure 4 Each panel shows the mean (7 SEM) result of analysing pairs of plasma samples from ten postmenopausal breast cancer patients taken before and during treatment with the aromatase inhibitor, anastrozole. The oestradiol was measured using direct and indirect assays. The direct assays were (b) Estradiol 33540; Beckman Coulter Access Immunoassay system, Fullerton, CA, USA, and (c) DSL-39100; 3rd Generation Estradiol Radioimmunoassay, Diagnostic Systems Laboratories Inc., Webster, TX, USA. The indirect assays were (a) extraction with diethyl ether followed by radioimmunoassay16 and (d) DSL-39100 kit after extraction of the plasma with diethyl ether.
in the same kit. Performance of this assay is then much closer to that seen with the optimised assay. This is an important deficit for oncologists to recognise if they wish to assess postmenopausal oestradiol levels: with routinely used assays it may not be possible to distinguish between pre- and postmenopausal levels, let alone the effect of aromatase inhibitors.
6.
7.
8.
References 1. Siiteri PK, MacDonald PC. Role of extraglandular estrogen in human endocrinology. In: Geiger SR, et al., editors. Handbook of physiology. Baltimore: Williams & Wilkins; 1973. p. 615–29. 2. Geisler J, Haynes B, Anker G, Dowsett M, Lonning PE. Influence of letrozole and anastrozole on total body aromatization and plasma estrogen levels in postmenopausal breast cancer patients evaluated in a randomized, crossover study. J Clin Oncol 2002;20:751–7. 3. Dunning AM, Dowsett M, Healey CS, et al. Polymorphisms associated with circulating sex hormone levels in postmenopausal women. J Natl Cancer Inst 2004; 96:936–45. 4. Key TJ, Phil D, ICRF Cancer Epidemiology Unit, University of Oxford, and affiliations of the Endogenous Hormones and Breast Cancer Collaborative Group. Endogenous sex hormones and breast cancer in postmenopausal women: reanalysis of nine prospective studies. J Natl Cancer Inst 2002;94:606–16. 5. Key TJ, Appleby PN, Reeves GK, of the Endogenous Hormones Breast Cancer Collaborative Group, et al. Body mass index, serum sex hormones, and breast cancer risk in
9.
10.
11.
12.
13.
postmenopausal women. J Natl Cancer Inst 2003;95: 1218–26. Dowsett M. Clinical trials with aromatase inhibitors for the prevention of breast cancer. In: Ingle JN, Dowsett M, editors. Advances in endocrine therapy of breast cancer. New York: Dekker; 2004. p. 189–201. Dowsett M, Stein RC, Coombes RC. Aromatization inhibition alone or in combination with GnRH agonists for the treatment of premenopausal breast cancer patients. J Steroid Biochem 1992;43:155–9. Klijn KG, Beex LV, Mauriac L, et al. Combined treatment with buserelin and tamoxifen in premenopausal metastatic breast cancer: a randomized study. J Natl Cancer Inst 2000; 92:903–11. Dowsett M, Haynes BP. Hormonal effects of aromatase inhibitors: focus on premenopausal effects and interaction with tamoxifen. J Steroid Biochem Mol Biol 2003;86: 255–63. Stein RC, Dowsett M, Hedley A, Coombes RC. The clinical and endocrine effects of 4-hydroxyandrostenedione alone and in combination with goserelin in premenopausal women with advanced breast cancer. Br J Cancer 1990;62: 679–83. Dowsett M, Doody D, Miall S, Howes A, English J, Coombes RC. Vorozole results in greater oestrogen suppression than formestane in postmenopausal women and when added to goserelin in premenopausal women with advanced breast cancer. Br Cancer Res Treat 1999;56: 25–34. Lonning PE, Johannessen DC, Lien EA, Ekse D, Fotsis T, Adlercreutz H. Influence of tamoxifen on sex hormones, gonadotrophins and sex hormone binding globulin in postmenopausal breast cancer patients. J Steroid Biochem Mol Biol 1995;52:491–6. Geisler J, King N, Anker G, et al. In vivo inhibition of aromatization by exemestane, a novel irreversible aromatase
ARTICLE IN PRESS The biology of steroid hormones and endocrine treatment of breast cancer inhibitor, in postmenopausal breast cancer patients. Clin Cancer Res 1998;4:2089–93. 14. Harper-Wynne C, Dowsett M, Ross G, et al. Effects of the aromatase inhibitor letrozole on normal breast epithelial cell proliferation and metabolic indices in postmenopausal women: a pilot study for breast cancer prevention. Cancer Epidem Biomarkers Prev 2002;11:614–21.
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15. Dowsett M, Folkerd E. Deficits in plasma oestradiol measurement in studies and management of breast cancer. Breast Cancer Res 2005;7:1–4. 16. Dowsett M, Goss PE, Powles TJ, et al. Use of the aromatase inhibitor 4-hydroxyandrostenedione in postmenopausal breast cancer: optimization of therapeutic dose and route. Cancer Res 1987;47:1957–61.
THE
BREAST www.elsevier.com/locate/breast
ORIGINAL ARTICLE
The biology of steroid hormones and endocrine treatment of breast cancer Mitch Dowsett, Elizabeth Folkerd, Deborah Doody, Ben Haynes Department of Biochemistry, Royal Marsden Hospital, London SW3 6JJ, UK
KEYWORDS Oestrogen; Breast cancer; Aromatase
Summary Recognition of the role of oestrogens in the stimulation of breast tumour growth has led to the development of several therapies based on endocrine intervention. Endocrine agents are currently used as a treatment for steroid receptor-positive breast cancer and more recently as a preventative measure in high-risk populations. Accurate quantification of resulting steroid hormonal profiles is essential to the understanding of the biological action and efficacy of these agents. In premenopausal women GnRH agonists suppress ovarian oestrogen synthesis and reduce oestradiol to close to postmenopausal levels. GnRH agonists used in combination with an aromatase inhibitor suppress levels even further. In contrast, tamoxifen can lead to markedly enhanced levels. In postmenopausal women aromatase inhibitors can achieve an almost complete inhibition of the aromatase enzyme. & 2005 Elsevier Ltd. All rights reserved.
Introduction The stimulation of breast cancer growth by oestrogens has provided a long established target for several well-tolerated treatments for steroid receptor-positive breast cancer and in recent years this has extended to their exploratory evaluation in the prevention context. In some cases the therapies used are specifically aimed at reducing oestrogen synthesis while in other cases the aim is to antagonise oestrogen action, but this can have secondary consequences for steroid hormone proCorresponding author. Tel.: +44 20 7808 2887;
fax: +44 20 7376 3918. E-mail address: [email protected] (M. Dowsett).
files as a result of feed-back mechanisms. An understanding of these effects is important for an appreciation of the mechanism of action of our most important endocrine agents and to allow their rational application in different clinical contexts.
Oestrogen synthesis In premenopausal women the large majority of plasma oestrogen synthesis is in the ovaries under the control of stimulatory effects of LH and FSH. Levels vary greatly during the menstrual cycle, ranging from 100 pmol/l or less in the very early follicular phase to values over 1000 pmol/l shortly
0960-9776/$ - see front matter & 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.breast.2005.08.019
ARTICLE IN PRESS The biology of steroid hormones and endocrine treatment of breast cancer prior to ovulation. Overall mean levels approximate to between 300 and 400 pmol/l. A small proportion of plasma oestrogens are derived from the peripheral synthesis by aromatase that is present in numerous non-glandular tissues, in particular subcutaneous fat. On average these peripheral sources make up less than 10% of the oestrogen in the circulation of premenopausal women, although it may be substantially greater in some obese women. Synthesis also occurs in the normal breast and in breast carcinomas and this source may be of particular relevance to the oestrogens that reach malignant breast cells. In postmenopausal women the ovaries cease their production of oestrogens as a result of the complete loss of primordial follicles. Oestrogens, however, continue to be synthesised by peripheral aromatisation from androgens that reach the circulation from both the adrenals and the ovaries. Peripheral aromatase activity increases with age such that this source of oestrogen approximately doubles in comparison to the same source in premenopausal women.1 Using the most sensitive assays (see later) plasma oestradiol levels approximate to mean levels of about 20 pmol/l in postmenopausal women.2,3 Oestradiol is the most potent of the nonconjugated oestrogens. It enters cells by free diffusion although its intracellular concentration is enhanced in those organs and tissues that express oestrogen receptors (ERs). The passage into the cells is impeded by the binding of oestradiol to albumin at low affinity and to sex hormone binding globulin (SHBG) at high affinity. Oestradiol is converted reversibly to oestrone, and oestrone itself is converted reversibly to oestrone sulphate. This latter conjugated oestrogen is not able to bind directly to the oestrogen receptor, but it may form an important reservoir for conversion to the primary oestrogens by the action of estrone sulphatase (Fig. 1).
androstenedione
testosterone
Steroids and the risk of breast cancer During the 1970s and 1980s many case-control studies were conducted to determine whether plasma steroid levels were related to a greater risk of developing breast cancer. However, the difficulty of avoiding bias in these studies resulted in a confused literature. More recently, a number of prospective studies have reported more consistent data and have culminated in an overview of nine such studies.4 This analysis revealed highly significant relationships between breast cancer risk and plasma oestradiol, oestrone, testosterone and androstenedione levels in postmenopausal women. A modest negative but significant relationship was found with SHBG. The relationship with oestradiol has been shown to largely explain the risk found between body mass index and breast cancer risk in postmenopausal women.5 These findings have led to the consideration that measurement of such hormone levels in postmenopausal women may enable them to be fitted into an algorithm of breast cancer risk.6 We have considered the possibility that plasma hormone levels might be subject to genetic controls and vary according to genetic polymorphisms in enzymes controlling their synthesis and metabolism.3 If so, this would provide indirect evidence for the involvement of these genes in breast cancer. We found that there was a highly significant relationship between plasma oestradiol and an even stronger relationship for plasma oestradiol/ testosterone ratio with the presence of a polymorphism in exon 10 of CYP19 (aromatase). We also established that there was a significant relationship between the plasma levels of SHBG and a polymorphism in the 50 -untranslated region of the SHBG gene. While these studies revealed highly statistically significant findings the proportion of the variability of the plasma hormone levels explained by the polymorphisms was small. At present it is unlikely that the assessment of these polymorphisms on their own would be useful in the evaluation of breast cancer risk and indeed they have not been shown to have a direct relationship with risk in relatively large-scale studies.3
SHBG
AROMATASE oestrone
453
oestradiol
oestrone sulphate
Figure 1 Steroids with major involvement in breast cancer. Testosterone and oestradiol bind with high affinity to sex hormone binding globulin (SHBG).
Premenopausal treatments Ovariectomy leads to a steroid hormone milieu that is very similar to that in postmenopausal women although oestrogen values may be marginally below those in postmenopausal women since the ovarian source of androgens is ablated by this operation. GnRH agonists are now a very popular means of
ARTICLE IN PRESS 454
M. Dowsett et al.
achieving a so-called medical ovariectomy. It can be seen in Fig. 2 that after 1 month’s treatment with 3.6 mg/month of goserelin in 20 patients with endometriosis, plasma oestradiol levels were reduced in all patients to mean values of 18 pmol/l.7 Thereafter, measurements made just prior to each of the monthly injections revealed that in virtually all cases the oestradiol levels remained below 100 pmol/l, but in many patients there was a minor degree of recovery above the 1-month levels, probably due to a low level of follicular oestrogenesis. In this series there was also one patient who showed a complete recovery of premenopausal levels. This particular patient showed a reaction around the injection site. Further studies revealed that this is a very uncommon but not unique example of such an absence of maintained suppression. When tamoxifen is administered to premenopausal women very high levels of oestradiol (sometimes as high as 3000 pmol/l or more) can occur.8 It is clear that despite this, tamoxifen remains an effective agent in premenopausal women, with similar activity to that achieved by ovariectomy or GnRH agonists. Calculations can be made on the competition between oestradiol with tamoxifen and its metabolites at the ER.9 These indicate that even in the presence of 1500 pmol/l of oestradiol 600
Plasma oestradiol (pmol/l)
500
400
300
200
100
0 Pre
1
2
3 4 Time (months)
5
6
Figure 2 Effect of goserelin (3.6 mg/month) on plasma oestradiol levels in 20 premenopausal endometriosis patients. Data summarised in Dowsett et al.7
(taken as a mean level in tamoxifen-treated premenopausal women) there is between 98% and 99% occupancy of the ER by tamoxifen such that the effective concentration of oestradiol on the receptor would be about 25 pmol/l. This would be consistent with a 10- to 20-fold reduction in oestradiol stimulation from baseline in such patients. When GnRH agonists and tamoxifen are given in combination there is no such increase in plasma oestradiol levels; rather the levels are similar to those seen with GnRH agonists alone and may be marginally less.8 In these circumstances tamoxifen would be expected to exert a full competition with oestradiol and therefore the combination could be expected to be more effective than either agent alone. This has been demonstrated to be the case in the small number of clinical trials conducted to date.8 Aromatase inhibitors are contra-indicated in premenopausal patients largely because of the expectation that they might cause cystic ovaries and lead to incomplete aromatase inhibition. This is rational and there is preclinical evidence of it, but the clinical experience to demonstrate this is in fact slight. We have reported a study of the triazole aromatase inhibitor YM511 in 30 premenopausal primary breast cancer patients who took part in a multi-centre, placebo-controlled, double-blind, randomised study in the 2 weeks prior to surgery.9 It was clear in this study that the expected increase in luteinising hormone (LH) and follicle-stimulating hormone (FSH) levels occurred in most patients and nearly all patients showed increases in testosterone and androstenedione as a result of this increased stimulation and aromatase inhibition. Effects on oestradiol levels were very variable with five of the on-treatment values in the 19 patients on YM511 being above the normal range of 100–900 pmol/l and five being below (Fig. 3). For oestrone, which is more dependent on peripheral synthesis, there were a greater number of patients with suppressed levels (Fig. 3). No suppression was found on the expression of the proliferation-associated nuclear antigen Ki67 in the tumours of these patients. This is in stark contrast to the suppression of Ki67 in the breast carcinomas of all but four of the 56 ERpositive, anastrozole-treated, postmenopausal patients over a similar 2-week period. These data therefore enhance the view that aromatase inhibitors are likely to have modest if any anti-tumour efficacy in premenopausal women. The use of aromatase inhibitors in combination with GnRH agonists is an area of increasing interest. We showed that the second generation aromatase inhibitor formestane suppressed the plasma levels
ARTICLE IN PRESS The biology of steroid hormones and endocrine treatment of breast cancer E1 (pmol/l)
E2 (pmol/l) 1500
600
1000
400
500
200
0
Nil
YM511 2w
0
Nil
YM511 2w
Figure 3 Comparison of plasma oestradiol and oestrone levels in premenopausal women after no treatment or 2 weeks’ treatment with YM511.9
of oestradiol consistently below 10 pmol/l in five premenopausal patients who were also treated with goserelin.10 We obtained similar data when determining the effect of the triazole aromatase inhibitor vorozole in combination with goserelin.11 Thus the combination of a GnRH agonist and an aromatase inhibitor appears to be a viable approach to obtaining near complete oestrogen deprivation in premenopausal women.
Postmenopausal treatments In postmenopausal women the endocrine effects of tamoxifen are much more modest than those in premenopausal women. There are increases in sex hormone binding globulin that would be expected to enhance the binding of plasma oestradiol and reduce its biological activity. The relationship between SHBG concentrations and residual free oestradiol is, however, relatively flat such that these changes, although to the advantage of the overall suppression of oestrogen stimulation of breast cancer, are probably only a minor contributor. There are increases in oestrone sulphate levels by about 20% and small decreases in plasma oestradiol levels (by about 10%), the latter probably being secondary to a fall in plasma testosterone due to a reduced ovarian secretion of this androgen.12 All of these changes in plasma steroids are probably of little importance in postmenopausal women treated with tamoxifen since it can be estimated that there is greater than 99.9% occupancy of ER by tamoxifen and its metabolites in this population.9 The third generation aromatase inhibitors achieve near complete inhibition of the enzyme in postmenopausal women. A cross-over study with 12
455
patients treated with both anastrozole and letrozole revealed that aromatase activity was suppressed to below 1% of baseline activity in all 12 patients by letrozole, but in only one patient on anastrozole in whom the mean residual levels were 2.7%.2 These latter levels were thus similar to those with exemestane at its clinical dose of 25 mg/day.13 In the same cross-over study of anastrozole/ letrozole all of the oestradiol levels were suppressed to below the detection limit of the assay (2.1 pmol/l) with letrozole, but not with anastrozole.2 However, the difference was not statistically significant. For oestrone and oestrone sulphate significant differences were seen with letrozole showing a greater suppression in both cases. Consideration of the clinical data between these two agents, however, suggests that these differences in pharmacological effectiveness are probably of only modest, if any, clinical significance. It is unusual when using highly sensitive oestradiol assays to find patients that do not show suppression to below or close to the detection of these assays. In one study14 we observed 2 out of 29 patients who did not show suppression at all after 3 months of letrozole treatment. In one of these patients drug levels were found to be low and compliance may have been poor. However, normal drug levels were seen in the other patient and an alternative explanation needs to be sought. It should be stressed, however, that in our experience less than 1% of postmenopausal patients do not show good suppression with modern inhibitors.
Problems of plasma oestradiol measurement in postmenopausal women We have recently published data illustrating the very poor performance of some plasma oestradiol assays that are in widespread routine use for the measurement of postmenopausal levels.15 In the main, these assays have been developed for the assessment of premenopausal levels and in association with ovulation induction schedules where very high levels are observed. Figure 4 summarises our observations and it is clear that the plasma levels seen with two such assays in which the oestrogen is not extracted from the plasma showed mean levels of about 100 pmol/l and very modest suppression with the aromatase inhibitor. This contrasts markedly with the mean levels seen when using a methodology that has been optimised for postmenopausal measurements. The major bias in the nonextraction assays is particularly clear when comparison is made with results obtained when oestradiol is extracted from the serum and assayed
ARTICLE IN PRESS 456
M. Dowsett et al.
Figure 4 Each panel shows the mean (7 SEM) result of analysing pairs of plasma samples from ten postmenopausal breast cancer patients taken before and during treatment with the aromatase inhibitor, anastrozole. The oestradiol was measured using direct and indirect assays. The direct assays were (b) Estradiol 33540; Beckman Coulter Access Immunoassay system, Fullerton, CA, USA, and (c) DSL-39100; 3rd Generation Estradiol Radioimmunoassay, Diagnostic Systems Laboratories Inc., Webster, TX, USA. The indirect assays were (a) extraction with diethyl ether followed by radioimmunoassay16 and (d) DSL-39100 kit after extraction of the plasma with diethyl ether.
in the same kit. Performance of this assay is then much closer to that seen with the optimised assay. This is an important deficit for oncologists to recognise if they wish to assess postmenopausal oestradiol levels: with routinely used assays it may not be possible to distinguish between pre- and postmenopausal levels, let alone the effect of aromatase inhibitors.
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