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

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 (E-1), estradiol (E-2) and estrone sulphate (EIS) in breast tumour tissue. Following incubation with [H-3]-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 E-2, and all three estrogen fractions were finally measured by the same highly sensitive and specific radioimmunoassay using estradiol-6-(O-carboxymethyl)-oximino-2-(2-[I-125]-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 E-1 and E-2 and 15-30% for E1S. The detection limit of this method was 4.3 fmol/g tissue for E-2, 19.8 fmol/g tissue for E-1 and 11.9 fmol/g E1S, respectively. Using tissue from locally advanced breast cancers (n = 14), we found median levels of E-1, E-2 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. (C) 2000 Elsevier Science Ltd. All rights reserved.