A summary of second-line randomized studies of aromatase inhibitors

The new generation of selective aromatase inhibitors (anastrozole, letrozole and exemestane) offer a significant efficacy and safety advantage over both older agents in this class (aminoglutethimide) and the progestins (megestrol acetate (MA)), as second-line treatment for postmenopausal women with advanced hormone-dependent breast cancer who have failed on tamoxifen therapy. Exemestane, a steroidal aromatase inhibitor, has been shown to have activity after failure with the non-steroidal aromatase inhibitors, anastrozole and letrozole, and could be used as third-line treatment. Although the newer aromatase inhibitors belong to the same class and appear, from indirect comparisons, to have similar efficacy compared with the older therapies, they have different pharmacokinetic and pharmacodynamic profiles, suggesting the potential for clinical differences. Compared with exemestane and letrozole, anastrozole shows greater selectivity for aromatase, as it lacks any evidence of an effect on adrenal steroidogenesis and has no androgenic effects. Therefore, it is clear that these agents should not be considered to be similar in all respects. In summary, the introduction of the aromatase inhibitors represents a significant step forward in the treatment of advanced breast cancer in postmenopausal women. Studies in the adjuvant setting will ultimately determine whether the differences in pharmacokinetics and phamacodynamics will be of clinical relevance.     

Endocrine approaches for the treatment of early and advanced breast cancer in postmenopausal women

Preventing clinical progression is the major treatment goal for both early and advanced breast cancer. For hormone-responsive cases (about 70% of the total), this can necessitate the use of sequential hormone therapies at various points during the patient's life. Newer hormonal therapies, such as the third-generation aromatase inhibitor anastrozole, are now competing with tamoxifen as first choice endocrine therapy in breast cancer. In addition, a further non-steroidal aromatase inhibitor letrozole has been shown to be beneficial when given at completion of 5 years adjuvant tamoxifen. In light of these new data, current treatment paradigms need to be reviewed. Already well established as second-line treatments for advanced breast cancer, the improved risk:benefit profiles of anastrozole and letrozole compared with tamoxifen mean that these agents are now also recognised alternative treatments in the first-line relapse setting. More recent studies demonstrate that anastrozole may also have an improved risk:benefit profile compared with tamoxifen when used as initial adjuvant therapy in early breast cancer. Anastrozole is also being evaluated as a preventative treatment in women at high risk of developing breast cancer. A new addition to the endocrine treatment armamentarium is the oestrogen receptor antagonist fulvestrant, which, unlike tamoxifen, has no agonist effects. Fulvestrant is at least as effective as anastrozole in the second-line treatment of advanced breast cancer, and provides similar benefits to tamoxifen when used as first-line therapy in patients with advanced, hormone receptor-positive tumours.     

Where do selective estrogen receptor modulators (SERMs) and aromatase inhibitors (AIs) now fit into breast cancer treatment algorithms?

The agents used for endocrine therapy in patients with breast cancer have changed markedly over the past decade. Tamoxifen remains the anti-oestrogen of choice, but could be replaced by the oestrogen receptor down-regulator ICI 182780 or by the fixed ring triphenylethylene arzoxifene (previously SERM III) soon. Whilst aminoglutethimide and 4-OH androstenedione were the aromatase inhibitors of choice, they have been replaced by non-steroidal (anastrozole and letrozole) and steroidal (exemestane) inhibitors of high potency and low side effect profile. Previously, often used treatments such as progestogens (megestrol acetate and medroxyprogesterone acetate) and androgens are now rarely used or confined to fourth or fifth line treatments. The LHRH agonist, goserelin, remains the treatment of choice for pre-menopausal patients with advanced breast cancer although recent randomised trials indicate a response, time to progression and survival advantage for the combination of goserelin and tamoxifen compared with goserelin alone.

The newer treatments have led to questions concerning the optimum sequence of agents to use in advanced breast cancer and as neo-adjuvant and adjuvant therapy in relation to surgery. Two trials of anastrozole compared with tamoxifen and one trial of letrozole compared with tamoxifen indicate that the new triazole aromatase inhibitors have a significant advantage over the anti-oestrogen with respect to time to progression and survival. Similarly, triazole aromatase inhibitors give faster and more complete responses compared with tamoxifen when used in post-menopausal women before surgery.

Major research questions remain with respect to the aromatase inhibitors used as adjuvant therapy. Anastrozole is being tested alone or in combination with tamoxifen compared with tamoxifen in the ‘so-called’ ATAC trial. Over 9000 patients have been randomised to this important study: the results will be available late-2001. A similar study comparing letrozole and tamoxifen started recently under the auspices of the Breast International Group. Importantly, this trial is also comparing the sequence of tamoxifen followed by letrozole (or vice versa). A similar trial of exemestane given after 2–3 years of tamoxifen compared with 5 years of tamoxifen is recruiting well as is a study comparing letrozole (or placebo) for 5 years after 5 years of adjuvant tamoxifen. These studies may show that aromatase inhibitors are superior to tamoxifen or that a sequence is preferable.

ICI 182780 causes complete oestrogen receptor down-regulation leading to a the lack of agonist activity of the drug. Two trials of ICI 182780 compared with anastrozole for advanced disease will report later this year and a comparison with tamoxifen next year. Arzoxifene (SERM III) is being tested against tamoxifen. These studies are likely to result in new anti-oestrogens being introduced into the clinic.

Most of our endocrine treatments deprived the tumour cell of oestradiol. In vitro experiments with MCF-7 cells indicate that tumour cells can adapt and then grow in response to low oestrogen concentrations in the tissue—culture medium. Importantly, the cells were shown to apoptose in response to high oestrogen concentrations. A recent clinical trial has demonstrated a high response rate to stilboestrol given after a median of four previous oestrogen depriving endocrine therapies. These data and the newer treatments available indicate a need to re-think our general approach to endocrine therapy and endocrine prevention.     

Aromatase inhibitors as adjuvant therapies in patients with breast cancer

There is increasing evidence that endocrine therapy has an important role in patients with oestrogen receptor positive breast cancer. Several large meta-analyses have reinforced the value of both ovarian ablation and tamoxifen in improving survival. Over the past decade, aromatase inhibitors have become the treatment of choice for second-line therapy of metastatic breast cancer, and the third generation inhibitors have now an established reputation for good patient tolerability. Early studies indicated that aminoglutethimide/hydrocortisone could benefit postmenopausal patients with primary breast cancer, and in 2001, the ATAC study showed that the third generation aromatase inhibitor, anastrozole, seemed superior to tamoxifen in that anastrozole-treated patients had a longer disease-free survival. Other studies will report on the relative merits of the steroidal inhibitor exemestane as well as non-steroidal letrozole. The exact duration and sequencing of treatment, together with the long-term effects on bone are at present, unknown.     

Inhibition of estradiol synthesis attenuates renal injury in male streptozotocin-induced diabetic rats

We previously showed that the male streptozotocin (STZ)-induced diabetic rat exhibits decreased circulating testosterone and increased estradiol levels. While supplementation with dihydrotestosterone is partially renoprotective, the aim of the present study was to examine whether inhibition of estradiol synthesis, by blocking the aromatization of testosterone to estradiol using an aromatase inhibitor, can also prevent diabetes-associated renal injury. The study was performed on male Sprague-Dawley nondiabetic, STZ-induced diabetic, and STZ-induced diabetic rats treated with 0.15 mg/kg of anastrozole, an aromatase inhibitor (Da) for 12 wk. Treatment with anastrozole reduced diabetes-associated increases in plasma estradiol by 39% and increased plasma testosterone levels by 187%. Anastrozole treatment also attenuated urine albumin excretion by 42%, glomerulosclerosis by 30%, tubulointerstitial fibrosis by 32%, along with a decrease in the density of renal cortical CD68-positive cells by 50%, and protein expression of transforming growth factor-β by 20%, collagen type IV by 29%, tumor necrosis factor-α by 28%, and interleukin-6 by 25%. Anastrozole also increased podocin protein expression by 18%. We conclude that blocking estradiol synthesis in male STZ-induced diabetic rats is renoprotective.     

Breast cancer tissue estrogens and their manipulation with aromatase inhibitors and inactivators

Despite the dramatic fall in plasma estrogen levels at menopause, only minor differences in breast tissue estrogen levels have been reported comparing pre- and postmenopausal women. Thus, postmenopausal breast tissue has the ability to maintain concentrations of estrone (E₁) and estradiol (E₂) that are 2–10- and 10–20-fold higher than the corresponding plasma estrogen levels. This finding may be explained by uptake of estrogens from the circulation and/or local estrogen production. Local aromatase activity in breast tissue seems to be of crucial importance for the local estrogen production in some patients while uptake from the circulation may be more important in other patients. Beside aromatase, breast tissue expresses estrogen sulfotransferase and sulfatase as well as dehydrogenase activity, allowing estrogen storage and release in the cells as well as conversions between estrone and estradiol. The activity of the enzyme network in breast cancer tissue is modified by a variety of factors like growth factors and cytokines. Aromatase inhibitors have been used for more than two decades in the treatment of postmenopausal metastatic breast cancer and are currently investigated in the adjuvant treatment and even prevention of breast cancer. Novel aromatase inhibitors and inactivators have been shown to suppress plasma estrogen levels effectively in postmenopausal breast cancer patients. However, knowledge about the influence of these drugs on estrogen levels in breast cancer tissue is limited. Using a novel HPLC-RIA method developed for the determination of breast tissue estrogen concentrations, we measured tissue E₁, E₂ and estrone sulfate (E₁S) levels in postmenopausal breast cancer patients before and during treatment with anastrozole. Our findings revealed high breast tumor tissue estrogen concentrations that were effectively decreased by anastrozole. While E₁S was the dominating estrogen fraction in the plasma, estradiol was the estrogen fraction with the highest concentration in tumor tissue. Moreover, plasma estrogen levels did not correlate with tissue estrogen concentrations. The overall experience with aromatase inhibitors and inactivators concerning their influences on breast tissue estrogen concentrations is summarized.     

Development and evolution of therapies targeted to the estrogen receptor for the treatment and prevention of breast cancer

This article describes the origins and evolution of "antiestrogenic" medicines for the treatment and prevention of breast cancer. Developing drugs that target the estrogen receptor (ER) either directly (tamoxifen) or indirectly (aromatase inhibitors) has improved the prognosis of breast cancer and significantly advanced healthcare. The development of the principles for treatment and the success of the concept, in practice, has become a model for molecular medicine and presaged the current testing of numerous targeted therapies for all forms of cancer. The translational research with tamoxifen to target the ER with the appropriate duration (5 years) of adjuvant therapy has contributed to the falling national death rates from breast cancer. Additionally, exploration of the endocrine pharmacology of tamoxifen and related nonsteroidal antiestrogen (e.g. keoxifene now known as raloxifene) resulted in the laboratory recognition of selective ER modulation and the translation of the concept to use raloxifene for the prevention of osteoporosis and breast cancer. However, the extensive evaluation of tamoxifen treatment revealed small but significant side effects such as endometrial cancer, blood clots and the development of acquired resistance. The solution was to develop drugs that targeted the aromatase enzyme specifically to prevent the conversion of androstenedione to estrone and subsequently estradiol. The successful translational research with the suicide inhibitor 4-hydroxyandrostenedione (known as formestane) pioneered the development of a range of oral aromatase inhibitors that are either suicide inhibitors (exemestane) or competitive inhibitors (letrozole and anastrozole) of the aromatase enzyme. Treatment with aromatase inhibitors is proving effective and is associated with reduction in the incidence of endometrial cancer and blood clots when compared with tamoxifen and there is also limited cross resistance so treatment can be sequential. Current clinical trials are addressing the value of aromatase inhibitors as chemopreventive agents for postmenopausal women.     

Plasma estrogen suppression with aromatase inhibitors evaluated by a novel, sensitive assay for estrone sulphate

Aromatase inhibition is a well-defined treatment option for postmenopausal breast cancer. Although several aromatase inhibitors such as aminoglutethimide, formestane, fadrozole have been found to inhibit in vivo aromatization by>85%, previous studies reported plasma estrogen levels to be sustained at approximately 20–50% of their control level during treatment with these drugs. The discrepancy could be due to lack of sensitivity or non-specific crossreactions in the radioimmunoassay (RIA) methods. Mean plasma levels of estrone (E₁) and estradiol (E₂) in postmenopausal women are approximately 80 and 20 pmol/l, respectively; on the contrary, mean plasma levels of the estrogen conjugate estrone sulphate (E₁S) are approximately 4–500 pmol/l. Most RIA methods for plasma E₂ and E₁ measurements have sensitivity limits in the range of 2–3 and 7–10 pmol/l, respectively; accordingly, the suppression of plasma estrogens by more than 80–90% will produce hormone values below the sensitivity limit of the method in many patients. Recently, we developed a new method to determine plasma E₁S. This assay has a sensitivity limit of 2.7 pmol/l. In theory, this method may allow the determination of plasma E₁S levels suppressed to less than 2% of control values in the majority of patients. Using this method, we found different aromatase inhibitors such as formestane, aminoglutethimide, formestane and aminoglutethimide administered in concert or anastrozole to suppress plasma E₁S levels down to 24, 13, 7 and 4%, respectively. The suppression of plasma E₁S evaluated with this method thus approaches the percentage aromatase inhibition measured with tracer studies.     

Are aromatase inhibitors superior to antiestrogens?

Aromatase inhibitors (AIs) have been in use to treat metastatic breast cancer for over 25 years. Recently potent and specific AIs have been introduced, which, because of their low toxicity profile, are being used in the adjuvant and neoadjuvant situation and also for the prevention of breast cancer. The two non-steroidal AIs, anastrozole and letrozole, and the steroidal AI, exemestane, have all shown superiority to tamoxifen as first-line treatment for advanced breast cancer. Interestingly, the oestrogen receptor downregulator, fulvestrant, was shown to be equivalent to anastrozole when compared as second-line therapy after the failure of tamoxifen. The first adjuvant AI trial began in 1996 and recruited over 9000 patients (ATAC trial). Anastrozole was compared with tamoxifen and a combination of the two drugs. There were no significant differences between tamoxifen and the combination. However, anastrozole showed about a 20% improvement in disease-free survival in ER+ disease compared with the other treatments. An overall survival analysis will be reported later this year. Two trials have compared 5 years of tamoxifen with 2–3 years of tamoxifen, followed by 2–3 years of AI (one trial (ITA) used anastrozole and another (intergroup) exemestane). Both trials show a disease-free advantage for the switch to AI. In another study (MA17) 5 years of tamoxifen was followed by a randomisation to letrozole or placebo and showed a significant disease-free advantage to the AI. Both letrozole and anastrozole show superiority to tamoxifen when used as a neoadjuvant therapy. Anastrozole significantly reduced contralateral breast cancer compared with tamoxifen, and this has led to two prevention trials: one in women at risk comparing anastrozole with placebo and the other after excision of DCIS comparing anastrozole with tamoxifen (IBIS II). The NCI Canada has also just initiated a trial of exemestane for prevention. Nearly all data available indicate that AIs are superior to tamoxifen. The important question is whether survival is improved when they are used as adjuvant therapy?     

Recent progress in development of aromatase inhibitors

In postmenopausal women with breast cancer, aromatase, which is the enzyme converting androstenedione to estrone and testosterone to estradiol, is the rate-limiting step in estrogen biosynthesis. The currently available aromatase inhibitor, aminogluethimide, effectively blocks estrogen production and produces tumor regressions in patients previously treated with tamoxifen. This drug, however, produces frequent side effects and blocks steroidogenic steps other than the aromatase enzyme. Thus, newer aromatase inhibitors with greater potency and specificity are under intense study. More than 20 such compounds have recently been developed. In several clinical trials, 4-hydroxyandrostenedione, given parenterally, has been highly active and specific for aromatase inhibition in patients with breast cancer. In two large recent studies, one-third of heavily pretreated women experienced objective tumor regression with this therapy. CGS 16949A, a newer agent, is also Phase II clinical trials. This compound is an imidazole derivative with nearly 1000-fold greater potency than aminoglutethimide. An initial Phase I study compared the potency of 0.6–16 mg daily in 12 postmenopausal women and found maximal suppression of urinary and plasma estrogens with 2 mg daily. The degree of inhibition was similar to that induced by aminoglutethimide or by surgical adrenalectomy. No CNS, hematologic or biochemical toxicity was observed. A larger Phase II study in 54 patients confirmed this high degree of potency of CGS since a plateau effect was observed at the 1.8, 2 and 4 mg daily doses. The endocrine effects were not absolutely specific as a blunting of ACTH-stimulated but not basal aldosterone levels were observed. This and other emerging aromatase inhibitors offer promise as pharmacologic methods to inhibit estrogen production specifically and without side effects.     

Aminoglutethimide and ketoconazole: historical perspectives and future prospects

Aminoglutethimide and ketoconazole, although originally developed as an anticonvulsant and antifungal agent respectively, have both been used to suppress steroid biosynthesis in patients with hormone-sensitive cancer. Aminoglutethimide inhibits several enzymes involved in the synthesis of corticosteroids as well as the aromatase enzyme which converts androgens to oestrogens. About one third of patients with breast cancer show objective improvement with aminoglutethimide, and it may also be of use in the treatment of adrenal carcinoma. However, its toxicity, and the need for concomitant cortisol replacement, severely limit its usefulness. Ketoconazole also inhibits several steroidogenic enzymes, notably C17,20-lyase, and has been used to treat carcinoma of the prostate. Again however, its toxicity and limited efficacy limit its value, although it may be useful in the treatment of certain endocrine conditions such as precocious puberty. Several aromatase inhibitors similar in structure to aminoglutethimide have been developed in an attempt to create more selective and efficient inhibitors. Some of these compounds have been tested in animals but none have as yet been subjected to clinical trials. Attempts to produce imidazole inhibitors of steroidogenesis are less advanced, although one compound (CGS 16949A) has been reported to be a more selective and potent aromatase inhibitor than aminoglutethimide. Selective and effective compounds could be of great value in the treatment of hormone-sensitive carcinoma.     

Mechanisms of the actions of aromatase inhibitors 4-hydroxyandrostenedione, fadrozole, and aminoglutethimide on aromatase in JEG-3 cell culture

Selective inhibition of estrogen production with aromatase inhibitors has been found to be an effective strategy for breast cancer treatment. Most studies have focused on inhibitor screening and in vitro kinetic analysis of aromatase inhibition using placental microsomes. In order to determine the effects of different inhibitors on aromatase in the whole cell, we have utilized the human choriocarcinoma cell line, JEG-3 in culture to compare and study three classes of aromatase inhibitors, 4-hydroxyandrostenedione, fadrozole (CGS 16949A), and aminoglutethimide. Fadrozole is the most potent competitive inhibitor and aminoglutethimide is the least potent among the three. However, stimulation of aromatase activity was found to occur when JEG-3 cells were preincubated with aminoglutethimide. In contrast, 4-OHA and fadrozole caused sustained inhibition of aromatase activity in both JEG-3 cells and placental microsomes, which was not reversed even after the removal of the inhibitors. 4-OHA bound irreversibly to the active site of aromatase and caused inactivation of the enzyme which followed pseudo-first order kinetics. However, 4-OHA appears to be metabolized rapidly in JEG-3 cells. Sustained inhibition of aromatase induced by fadrozole occurs by a different mechanism. Although fadrozole bound tightly to aromatase at a site distinct from the steroid binding site, the inhibition of aromatase activity by fadrozole does not involve a reactive process. None of the inhibitors stimulated aromatase mRNA synthesis in JEG-3 cells during 8 h treatment. The stimulation of aromatase activity by AG appeared to be due to stabilization of aromatase protein. According to these results, 4-OHA and fadrozole would be expected to be more beneficial in the treatment of breast cancer patients than AG. The increase in aromatase activity by AG may counteract its therapeutic effect and might be partially responsible for relapse of breast cancer patients from this treatment.     

Inhibition of aromatase: insights from recent studies

Aromatase is the rate limiting enzyme that catalyzes the conversion of androgens to estrogens. Blockade of this step allows treatment of diseases that are dependent upon estrogen. Over the past two decades, highly potent and specific aromatase inhibitors have been developed which block total body aromatization by over 99%. An important recent question is whether aromatase inhibitors are superior to the antiestrogens for treatment of hormone-dependent breast cancer. The third generation aromatase inhibitors have been compared to tamoxifen for the treatment of breast cancer in the advanced, adjuvant, and neoadjuvant settings. All of these studies suggest the superiority of aromatase inhibitors over tamoxifen. The mechanism responsible for the superiority of the aromatase inhibitors relates to the estrogen agonistic effects of tamoxifen. During exposure to estrogen deprived conditions and to tamoxifen, breast cancer cells adapt and upregulate the MAP kinase and PI-3 kinase pathways. These growth factor signaling pathways potentiate the estrogen agonistic properties of tamoxifen. Data from a large adjuvant therapy trial (ATAC trial) provide evidence that the aromatase inhibitors may also be superior for breast cancer prevention. The mechanism for superiority in this setting probably relates to the genotoxic effects of estradiol metabolites. The aromatase inhibitors may be also useful for the treatment of endometriosis and for ovulation induction as evidenced by preliminary data. The recent advances in development of the aromatase inhibitors clearly demonstrate the utility of these agents for treatment of breast cancer and potentially for other indications.     

Aromatase and its inhibitors

Inhibitors of aromatase (estrogen synthetase) have been developed as treatment for postmenopausal breast cancer. Both steroidal substrate analogs, type I inhibitors, which inactivate the enzyme and non-steroidal competitive reversible, type II inhibitors, are now available. 4-hydroxyandrostenedione (4-OHA), the first selective aromatase inhibitor, has been shown to reduce serum estrogen concentrations and cause complete and partial responses in approximately 25% of patients with hormone responsive disease who have relapsed from previous endocrine treatment. Letrozole (CGS 20, 269) and anastrozole (ZN 1033) have been recently approved for treatment. Both suppress serum estrogen levels to the limit of assay detection. Letrozole has been shown to be significantly superior to megace in overall response rates and time to treatment failure, whereas anastrozole was found to improve survival in comparison to megace. Both were better tolerated than the latter. The potential of aromatase within the breast as a significant source of estrogen mediating tumor proliferation and which might determine the outcome of inhibitor treatment was explored. Using immunocytochemistry and in situ hybridization, aromatase and mRNA_arom was detected mainly in the epithelial cells of the terminal ductal lobular units (TDLU) of the normal breast and also in breast tumor epithelial cells as well as some stromal cells. Increase in proliferation, measured by increased thymidine incorporation into DNA and by PCNA immunostaining in response to testosterone was observed in histocultures of breast cancer samples. This effect could be inhibited by 4-OHA and implies that intratumoral aromatase has functional significance. An intratumoral aromatase model in the ovariectomized nude mouse was developed which simulated the hormone responsive postmenopausal breast cancer patient. This model also allows evaluation of the efficacy of aromatase inhibitors and antiestrogens in tumors of estrogen receptor positive, human breast carcinoma cells transfected with the human aromatase gene. Thus, the cells synthesized estrogen which stimulated tumor formation. Both aromatase inhibitors and antiestrogens were effective in suppressing tumor growth in this model. However, letrozole was more effective than tamoxifen. When the aromatase inhibitors were combined with tamoxifen, tumor growth was suppressed to about the same extent as with the aromatase inhibitors alone. Thus, there was no additive or synergistic effects of combining tamoxifen with aromatase inhibitors. This suggests that sequential treatment with these agents is likely to be more beneficial to the patient in terms of longer response to treatment.     

Local endocrine effects of aromatase inhibitors within the breast

To determine the effects of aromatase inhibitors on oestrogen uptake, in situ aromatase activity and endogenous oestrogens in the breast, postmenopausal women with large primary ER-rich breast cancers have been treated neoadjuvantly for 3 months with either letrozole (2.5 or 10 mg daily) or anastrozole (1 or 10 mg daily) or exemestane (25 mg daily). Patients were given an infusion of ³H-androstenedione and ¹⁴C-oestrone for 18 h before and at the end of the study period. Blood, tumour and non-malignant breast were taken immediately after each infusion; oestrogens were extracted and purified. Tumour volume was measured before and during treatment at monthly intervals so that endocrinological changes could be related to clinical response. Treatment with each of the aromatase inhibitors was associated with a profound reduction in peripheral aromatase (as monitored by the level of plasma ³H-oestrone). There was no consistent effect on uptake of radioactively labelled oestrogen into breast tumours but a tendency for levels to increase after treatment in non-malignant breast. Conversely, therapy was associated with a marked inhibition of in situ oestrogen synthesis in both tumour and non-malignant breast (in occasional tissues, inhibitors appeared to be less effective but the effect was not related to clinical or pathological responses). Similar decreases were apparent in endogenous levels of oestrone and oestradiol. The absence of in situ aromatase activity tended to be associated with lack of clinical response to aromatase inhibition but the relationship was not absolute, limiting the utility of measurements of tumour aromatase as a predictive indices. Ex vivo studies of tissue aromatase indicated that such measurements consistently underestimate the inhibitory potential of reversible non-steroidal agents (and occasionally paradoxical in vitro increases in aromatase activity were seen with treatment). However, in situ assays demonstrate that new aromatase inhibitors such as anastrozole, exemestane and letrozole have profound effects on the local endocrinology within the postmenopausal breast, these being compatible with the clinico-pathological changes which occur with treatment.     

The importance of local synthesis of estrogen within the breast

Tumor aromatase has been correlated with clinical response to treatment with aminoglutethimide in patients with estrogen receptor-positive advanced breast cancer. There was a significant positive relationship between aromatase status and likelihood of response to therapy, none of five patients with aromatase-negative tumors responding compared with 11 of 18 having aromatase-positive cancers. Measurement of aromatase in sequential biopsies of large primary tumors before and during treatment with aminoglutethimide-hydrocortisone showed a marked but paradoxical rise in activity following therapy. Assays of aromatase in adipose tissue from the different quadrants of mastectomy specimens from patients with breast cancer indicate that activity was always higher in quadrants associated with tumor as compared with non-involved quadrants. These results emphasize the importance of local estrogen synthesis within the breast in terms of both the natural history and behavior of breast cancers.     

Role of steroid sulfatase in local formation of estrogen in post-menopausal breast cancer patients

More than two-thirds of breast cancers occur in post-menopausal women, and depend on the estrogens for their proliferation and survival. For the treatment of estrogen-dependent breast cancers, two major treatment options are now available. One is selective estrogen receptor modulator (SERM) such as Tamoxifen and another is aromatase inhibitor such as Anastrozole, Letrozole and Exemestane, which reduce local in situ formation of estrogens. Although these therapies are clinically active for advanced and early breast cancers, de novo and/or acquired resistance to SERM and/or aromatase inhibitors are also clinical problem. Recent studies suggest that local formation of estrogens in the breast tumors is more important than circulating estrogen in plasma for the growth and survival of estrogen-dependent breast cancer in post-menopausal women. The rationale for the importance of local formation of estrogens is based on the following evidences. Estradiol (E2) levels in breast tumors are equivalent to those of pre-menopausal patients, although plasma E2 levels are 50-fold lower after menopause. E2 concentrations in breast tumors of post-menopausal women are 10–40 times higher than serum level. Biosynthesis of estrogens in breast tumors tissues occurs via two major different routes, one is aromatase pathway and another is steroid-sulfatase (STS) pathway. Whereas many studies has been reported about aromatase inhibitor and its clinical trial results in breast cancer patients, limited information are available regarding to other estrogen regulating enzymes including STS, its role in breast tumors and STS inhibitors. STS is the enzyme that hydrolyses estrone 3-sulfate (E₁S) and dehydroepiandrosterone-sulfate (DHEA-S) to their active un-sulfoconjugated forms, thereby stimulating the growth and survival of estrogen-dependent breast tumors. It has been well known that E₁S level are much higher than E2 level both in plasma and tumor of post-menopausal patients. Recent reports show that more than 80% of breast tumors are stained with anti-STS antibody and the expression of STS is an independent prognostic factor in breast cancer. Taking these findings into consideration, local formation of estrogens could be partially synthesized from large amount of E₁S by STS, which exist in breast cancer. On the other hand, aromatase localizes in stroma and adipocyte surrounding breast cancer. Furthermore, since estrogen formation from E₁S and DHEA-S (STS pathway) cannot be blocked by aromatase inhibitors, STS is thought to be a new molecular target for the treatment of estrogen-dependent tumor post-SERM and/or aromatase inhibitors. In this symposium, these recent rationale for the importance of STS in post-menopausal breast cancer patients is reviewed as well as STS inhibitor.     

Second line endocrine treatment of postmenopausal advanced breast cancer. Preliminary endocrine results of a 5-ARM randomized phase II trial of medium vs low dose aminoglutethimide, with or without hydrocortisone vs hydrocortisone alone (EORTC 10861)

The supposed mechanism of action of aminoglutethimide (AG), medical adrenalectomy, has been challenged. AG is now considered to act as an inhibitor of the aromatization of mainly adrenal androgens to estrogens in peripheral tissues and/or breast cancer itself. To further establish the AG dose required to sufficiently reduce estrogen levels in plasma and the possible role of hydrocortisone (HC) in combination with AG or by itself, postmenopausal advanced breast cancer patients received AG low (125 mg bid) or medium (250 mg bid) dose alone or combined with HC (20 mg bid) or HC alone (20 mg bid). Preliminary hormonal data show a similar reduction of serum estrone and estrone sulphate by at least some 50% at 8 wk in all treatment groups. At 6 months these effects persist except for patients treated with HC alone. In the latter a normalization of estrone levels is observed with effective suppression of adrenal androgen precursors, suggesting increased aromatase activity with prolonged glucocorticoid treatment.     

Intracellular aromatase and its relevance to the pharmacological efficacy of aromatase inhibitors

An important feature of the pharmacological profile of aromatase inhibitors is the ability of the various inhibitors to inhibit intracellular aromatase. It is now well documented that a large proportion of breast tumors express their own aromatase. This intratumoral aromatase produces estrogen in situ and therefore may contribute significantly to the amount of estrogen to which the cell is exposed. Thus it is not only important that aromatase inhibitors potently inhibit the peripheral production of estrogen and eliminate the external supply of estrogen to the tumor cell, but that they in addition potently inhibit intratumoral aromatase and prevent the tumor cell from making its own estrogen within the cell. To study the inhibition of intracellular aromatase we have compared the aromatase-inhibiting potency of the non-steroidal aromatase inhibitors, letrozole, anastrozole and fadrozole in a variety of model cellular endocrine and tumor systems which contain aromatase. We have used hamsters ovarian tissue fragments, adipose tissue fibroblasts from normal human breast, the MCF-7Ca human breast cancer cell line transfected with the human aromatase gene and the JEG-3 human choriocarcinoma cell line. Although letrozole and anastrozole are approximately equipotent in a cell-free aromatase system (human placental microsomes), letrozole is consistently 10–30 times more potent than anastrozole in inhibiting intracellular aromatase in intact rodent cells, normal human adipose fibroblasts and human cancer cell lines. Whether these differences between letrozole and anastrozole are seen in the clinical setting will have to await the results of clinical trials which are currently in progress.     

Postmenopausal estrogen synthesis and metabolism: alterations caused by aromatase inhibitors used for the treatment of breast cancer

Inhibition of postmenopausal estrogen production by aromatase inhibitors is an established drug treatment modality for postmenopausal breast cancer. In this article postmenopausal estrogen disposition and the alterations caused by treatment with aromatase inhibitors are reviewed. Recent investigations have challenged the hypothesis that aromatization of androstenedione into estrone is the sole production pathway for estrogens in postmenopausal women. The finding that estrogens persist in the plasma of patients receiving aminoglutethimide treatment despite a near total inhibition of the aromatase enzyme suggests that alternative pathways for estrogen synthesis exist. While nonspecific actions of aromatase inhibitors may be disadvantageous, certain effects may also be beneficial. Recent findings that aminoglutethimide may induce estrone sulfate metabolism questions whether this "prototype" aromatase inhibitor might have a dual mechanism of action. The importance of investigating the possible influence of different aromatase inhibitors on all components of estrogen disposition is considered.