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

Abstract

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.

Introduction

Plasma estradiol (E₂) levels decrease at menopause by about 90% and one could expect that breast tissue E₂ levels would decrease simultaneously. However, several investigators have confirmed that breast tissue E₂ levels are maintained at nearly the same level as seen in premenopausal women. Thus, breast cancer tissue E₂ levels are about 10–20-fold higher and breast tissue estrone (E₁) levels 2–10-fold higher than their corresponding plasma levels [1], [2], [3], [4]. This observation may be explained by local synthesis and/or active estrogen uptake from the circulation as well as altered estrogen metabolism. Moreover, the ratios of the major estrogen fractions seem to differ significantly between the circulation and breast tissue. While estrone sulfate (E₁S) is the dominating estrogen fraction in the plasma followed by E₁ and E₂, E₂ is the major estrogen fraction in postmenopausal breast cancer tissue followed by E₁ and E₁S.

Estrogen deprivation is an effective approach for the treatment of hormone sensitive breast cancer. Thus, plasma estrogen measurements have been performed to evaluate treatment options like castration in premenopausal women and treatment with aromatase inhibitors or inactivators in postmenopausal women. However, the major aim of estrogen depriving therapies is to decrease the estrogen concentrations in the malignant tissue and in non-malignant tissues surrounding the tumor. Moreover, plasma estrogen levels do not reflect tissue estrogen levels. Thus, there is an increasing interest in tumor tissue estrogen levels.

The main reasons for our limited knowledge about tumor tissue estrogen levels are the methodological problems connected to tissue estrogen measurements. Firstly, breast tissue is well-known to be a difficult type of tissue to work with for technical reasons, as it may contain significant amounts of connective tissue, fat and sometimes necrotic tissue. Secondly, a large variety of steroid hormones that may possibly cross-react in the detection assays are suggested to be present in breast tissue simultaneously. This risk of cross-reacting metabolites is substantially increased during treatment with steroidal drugs used for the treatment of breast cancer. Despite these methodological problems, several investigators have measured normal breast and breast cancer tissue estrogen levels using a variety of methods [2], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. The results of these studies are given in Table 1.

To more precisely measure tissue estrogen levels, we have recently established a novel, highly sensitive and specific HPLC-RIA especially designed for the simultaneous detection of breast tissue levels of E₁, E₂ and E₁S [14]. Briefly, tissue homogenates are incubated with ³H-labeled estrogens (E₁, E₂ and E₁S) as recovery controls and crude fractions are separated by ether extraction. The E₁S fraction is hydrolyzed with sulfatase followed by elution on a Sephadex LH-20 column. A HPLC system was used to purify the individual estrogen fractions prior to RIA analysis. E₁ and E₁S are converted into E₂, and all three estrogen fractions are finally measured by the same highly sensitive and specific RIA using estradiol-6-carboxy-methyloxime-[2-${}^{125}\text{I}$]-iodohistamine as a ligand. Final estrogen values are corrected for the amount of tissue used in each individual sample (wet weight) as well as for the recovery of the amount of [³H]-hormone added as internal standard. The detection limits for breast tissue levels of E₂, E₁ and E₁S are 4.3, 19.8, and 11.9 fmol/g tissue, respectively. We have recently measured E₁, E₂ and E₁S concentrations in the plasma and breast cancer tissue of 12 untreated postmenopausal women with advanced breast cancer (Fig. 1) and following treatment with anastrozole [9]. Thus, the described method proved to be suitable for the detection of very low estrogen levels, as expected in samples collected during treatment with the highly potent third generation aromatase inhibitors and inactivators.