A novel HPLC-RIA method for the simultaneous detection of estrone, estradiol and estrone sulphate levels in breast cancer tissue

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Abstract

Estrogen deprivation is an effective approach for treatment of hormone sensitive breast cancer. While much is known about plasma estrogen levels with respect to castration in premenopausal women and use of aromatase inhibitors in postmenopausal women, currently there is increasing interest in intra-tumour estrogen production. However, knowledge about alterations in intra-tumour estrogen levels is limited, mainly due to methodological problems with measurements of estrogen fractions in tissue samples. Here we describe a new method for simultaneous measurement of the three main estrogen fractions, estrone (E1), estradiol (E2) and estrone sulphate (E1S) in breast tumour tissue. Following incubation with 3H-labelled estrogen standards, crude fractions were separated by ether extraction. The E1S fraction was hydrolysed with sulphatase followed by eluation on a Sephadex column. High pressure liquid chromatography (HPLC) was used to purify the individual estrogen fractions prior to RIA analysis. Estrone and E1S were converted into E2, and all three estrogen fractions were finally measured by the same highly sensitive and specific radioimmunoassay using estradiol-6-(O-carboxymethyl)-oximino-2-(2-125I-iodo-histamine) as a ligand. Although several purification steps were used, the internal recovery values for tritiated estrogens were found to be 25–50% for E1 and E2 and 15–30% for E1S. The detection limit of this method was 4.3 fmol/g tissue for E2, 19.8 fmol/g tissue for E1 and 11.9 fmol/g E1S, respectively. Using tissue from locally advanced breast cancers (n = 14), we found median levels of E1, E2 and E1S to be 283.8 fmol/g tissue (range 19.8–547.5), 554.1 fmol/g (9.5–3024.2) and 209.4 fmol/g (11.9–753.4), respectively. The method described here is a promising tool to study intra-tumour estrogen fractions in breast tissue biopsies.

Introduction

Estrogens are known to stimulate growth of neoplasia in hormone sensitive tissues like the breast and the endometrium [1]. Thus, for many years scientific studies have measured plasma estrogen levels to evaluate the biochemical effects of different treatment options like aromatase inhibitors. However, plasma estrogen levels do not necessarily reflect tissue estrogen concentrations. It is well-established that breast cancer cells [2] as well as connective tissue cells within tumours [3] express the aromatase enzyme, responsible for the final step in estrogen synthesis, and current evidence suggests local estrogen production to be a major pathway contributing to intra-tumour estrogen levels [4]. Moreover, while the aromatase gene in tumour cells and peripheral tissues is similar, local aromatase could be stimulated by growth factors and interleukins locally expressed in tumour tissue [5], [6]. Finally, results from animal studies suggest concentration-dependent uptake of circulating estrogens to the tissue [7]. Thus, it is highly important to evaluate not only alterations in plasma but also intra-tumour estrogen levels in response to endocrine therapy.

Recent studies have revealed third generation aromatase inhibitors to inhibit total body aromatization by 97–98% [8], [9], [10] and to suppress plasma estrogen levels by more than 90%. While several studies have evaluated estrogen concentrations in normal and malignant breast tissue of postmenopausal women [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], only a few studies have determined tissue estrogen levels in patients during treatment with aromatase inhibitors [24], [25] due to technical problems in measuring tissue estrogens in the low range.

Here, we describe a sensitive method for the simultaneous measurement of the main estrogen fractions, estrone (E1), estradiol (E2) and estrone sulphate (E1S), in malignant breast tissue. The method described here involves HPLC purification of estrogen fractions and conversion of E1 and E1S to E2 followed by radioimmunoassay (RIA) analysis using an iodinated tracer.

Section snippets

Materials and methods

Radiolabelled estrogens, [2,4,6,7,-3H]E1 (101 Ci/mmol), [2,4,6,7,16,17-3H]E2 (170 Ci/mmol) and [6,7,-3H]E1S (60 Ci/mmol) for recovery determinations were obtained from DuPont NEN (Boston, MA) and estradiol-6-(O-carboxymethyl)-oximino-2-(2-125I-iodo-histamine) (2000 Ci/mmol) from Amersham International (Little Chalfont, U.K.). Sephadex LH-20 was obtained from Pharmacia (Uppsala, Sweden), sulphatase (S-9754) from Sigma Chemical Co. (London, U.K.), and the E2 antibody (ER 150, Sorin Biomedica

Results

The dectection limits of this method are 4.3 fmol/g tissue for E2, 19.8 fmol/g tissue for E1 and 11.9 fmol/g tissue for E1S, respectively. The overall recovery through all purification steps was about 25–50% for [3H]E1 and [3H]E2 and 15–30% for [3H]E1S. A typical eluation curve for radiolabelled estrogens following separation with HPLC is shown in Fig. 2.

The intra-assay coefficient of variation (CV) was determined by repeated analysis of 8–10 parallel samples obtained from a single primary

Discussion

Homogenization of breast tumour tissue may be difficult due to its varying consistency, sometimes containing a high proportion of connective tissue. Following evaluation of several methods, we found the combined use of ultra-turrax and ultrasound to be the most effective procedure obtaining the highest recovery of internal [3H]-labelled estrogen standards. The addition of other steps, like ultracentrifugation followed by resuspension, did not increase the percentage of recovery. This finding

Acknowledgments

This work was supported by grants from the Norwegian Cancer Society. We are grateful to Mr. D. Ekse for his skilful technical assistance, Dr. B. Lindtjorn (Department of Surgery, Haukeland University Hospital) for providing the tumour tissue samples, Mr. P. Skulstad (Department of Biochemical Endocrinology, Haukeland University Hospital) and Mr. J. Schneede (Department of Biochemistry, Section for Pharmacology, Haukeland University Hospital) for valuable discussion. J. Geisler is the recipient

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