Exploring breast cancer estrogen disposition: the basis for endocrine manipulation.

Although normal breast tissue and breast cancer estrogens are known to be elevated compared with plasma estrogen levels, the mechanism behind this phenomenon has been an issue of debate for 2 decades. If local estrogen aromatization were to be confirmed as the main estrogen source in breast cancer tissue, tissue-specific inhibition of estrogen production, avoiding systemic side effects, would become a potentially attractive option for breast cancer treatment and prevention. Based on recent results from our groups exploring tissue estrogens, together with estrogen-synthesizing and estrogen-regulated gene expression levels, we propose a new model to explain elevated breast tissue estrogen levels. Although local estrogen production may be important, the local contribution is overruled by rapid plasma-to-tissue equilibration, including active uptake of circulating estrogens or enhanced tissue binding. As for breast cancer tissue levels, elevated levels of estradiol may be explained to a large extent by estrogen receptor binding and local conversion of estrone into estradiol. This model indicates that effective suppression of benign and malignant tissue estrogens as a treatment for ER+ breast cancer requires systemic suppression and will not be markedly affected by local enzyme targeting.

Recent data on intratumor estrogens in breast cancer.

The contribution of local synthesis versus circulatory delivery of normal breast as well as breast cancer tissue estrogens has remained a controversial area for decades. Novel data on tissue estrogen levels confirm a positive normal breast tissue to plasma concentration gradient for estrone, and to a smaller extent estradiol. Remarkably, this gradient is similar for pre- and post-menopausal women. Together with pharmacokinetic data on estrogen disposition, these findings suggest plasma and breast tissue estrogens to rapidly equilibrate, with circulating estrogens being a major contributor to breast tissue estrone levels. A likely explanation to the concentration gradient could be the fact that non-polar estrogens easily dissolve into tissue fat compartments as compared to plasma. While intratumor estrone levels are low as compared to benign tissue concentrations, intratumor estradiol is elevated in ER+ tumors. The correlation between intratumor estradiol levels and expression levels of dehydrogenases reducing estrone into estradiol but also intratumor ER concentrations are consistent with intratumor estrogen activation but also a scavenger effect of the ER.

Intratumoral estrogen disposition in breast cancer.

PURPOSE: The concentration of estradiol (E(2)) in breast tumors is significantly higher than that in plasma, particularly in postmenopausal women. The contribution of local E(2) synthesis versus uptake of E(2) from the circulation is controversial. Our aim was to identify possible determinants of intratumoral E(2) levels in breast cancer patients. EXPERIMENTAL DESIGN: The expression of genes involved in estrogen synthesis, metabolism, and signaling was measured in 34 matched samples of breast tumor and normal breast tissue, and their correlation with estrogen concentrations assessed. RESULTS: ESR1 (9.1-fold; P < 0.001) and HSD17B7 (3.5-fold; P < 0.001) were upregulated in ER(+) tumors compared with normal tissues, whereas STS (0.34-fold; P < 0.001) and HSD17B5 (0.23-fold; P < 0.001) were downregulated. Intratumoral E(2) levels showed a strong positive correlation with ESR1 expression in all patients (Spearman r = 0.55, P < 0.001) and among the subgroups of postmenopausal (r = 0.76, P < 0.001; n = 23) and postmenopausal ER(+) patients (r = 0.59, P = 0.013; n = 17). HSD17B7 expression showed a significant positive correlation (r = 0.59, P < 0.001) whereas HSD17B2 (r = -0.46, P = 0.0057) and HSD17B12 (r = -0.45, P = 0.0076) showed significant negative correlations with intratumoral E(2) in all patients. Intratumoral E(2) revealed no correlation to CYP19, STS, and HSD17B1 expression. Multivariate models comprising ESR1 and plasma E(2) predicted between 50% and 70% of intratumoral E(2) variability. CONCLUSION: Uptake due to binding to the ER, rather than intratumoral estrogen synthesis by aromatase or sulfatase, is the single most important correlate and a probable determinant of intratumoral E(2). An increased expression of HSD17B7 may explain the increased ratio of E(2) to estrone (E(1)) in breast tumors compared with normal tissue.

Breast cancer aromatase expression evaluated by the novel antibody 677: correlations to intra-tumor estrogen levels and hormone receptor status.

Breast cancer tissue estrogen levels on an average exceed plasma as well as benign breast tissue levels. To evaluate the contribution of intra-tumor aromatization to individual tumor estrogen levels (estradiol, E2; estrone, E1; estrone sulfate, E1S), breast cancer tissue sections obtained during mastectomy in 28 postmenopausal breast cancer patients were stained for aromatase protein expression using the aromatase antibody 677. The findings were correlated to intra-tumor estrogen levels determined with a highly sensitive HPLC-RIA. Staining with 677 alone (irrespective of the hormone receptor status) revealed no difference in tumor E2 levels comparing 677+ versus 677- tumors, although a non-significant trend towards higher tumor E1 and E1S levels was observed in 677+ breast cancers. In contrast, tumor levels of E(2) were significantly higher in ER+ tumors compared to ER- tumors (P<0.001) and to benign breast tissue from the same breast (P<0.001). Analysing the additional effect of positive staining with the aromatase antibody 677 on tumor estrogen levels in the subgroup of ER+ tumors, revealed significantly higher tumor levels of E2 (mean level of 544.7 versus 197.1 fmol/g tissue) as well as a non-significant trend concerning tumor E1 (mean level of 296.9 versus 102.1 fmol/g tissue). The mean tumor tissue E1S level was observed somewhat lower in ER+677+ (103.5 fmol/g) versus ER+677- tumors (190.1 fmol/g). In the subgroup of ER+PgR+ tumors, tissue levels of E2 were also found to be significantly higher among 677+ compared to 677- tumors: 873.2 fmol/g (95% CI 395.9-1925.6) versus 217.9 fmol/g (95% CI 88.8-534.9) (P=0.015). In conclusion, our results indicate a moderate effect of aromatase enzyme expression evaluated by IHC using the antibody 677 on intra-tumor estrogen levels among ER+ breast cancers. A substantial interindividual variation in the ratios between the individual estrogen fractions suggests additional effects, like alterations in other enzymes to be involved in the intra-tumor estrogen homeostasis.

An optimised, highly sensitive radioimmunoassay for the simultaneous measurement of estrone, estradiol and estrone sulfate in the ultra-low range in human plasma samples.

Following the introduction of potent aromatase inhibitors for the treatment of breast cancer patients, highly sensitive methods have become mandatory to evaluate the influence of these drugs on plasma estrogen levels. Commercially available kits for estrogen measurements are not suitable for these kinds of evaluations due to their detection limits that are close to baseline estrogen levels in postmenopausal women. We describe here an optimised radioimmunoassay suitable for the simultaneous measurement of plasma estrone (E1), estradiol (E2) and estrone sulfate (E1S) levels in the ultra-low range. Following incubation with [3H]-labelled estrogens as internal standards, crude estrogen fractions were separated by ether extraction. The E1S fraction was hydrolysed with sulfatase followed by eluation on a Sephadex column. Free estrogens (E1, E2) were separated by chromatography (LH-20). Estrone and E1S (following hydrolysis) were converted into E2, and each estrogen fraction was measured by the same highly sensitive and specific radioimmunoassay using estradiol-6-(O-carboxymethyl)-oximino-2-(2-[125 I]-iodo-histamine) as ligand. Although several purification steps were involved, the internal recovery values for tritiated estrogens were found to be 88%, 90%, and 49% for E1, E2 and E1S, respectively. The intra-assay coefficient of variation was <5% for all recovery measurements. The detection limits were calculated following repeated blank measurements and found to be 1.14 pmol/L for E1, 0.67 pmol/L for E2, and 0.55 pmol/L for E1S, respectively. The intra-assay coefficient of variation (CV) was found to be 3.4% for E1, 5.1% for E2 and 6.1% for E1S, while the inter-assay CV was 13.6%, 7.6% and 7.5% for E1, E2, and E1S, respectively. Considering normal plasma levels for E2 (15 pmol/L), E1 (80 pmol/L) and E1S (400 pmol/L) in postmenopausal women, the method allows theoretically to detect suppression of plasma E2, E1 and E1S levels by 95.5%, 98.6% and 99.9% when starting from average, normal postmenopausal levels. Thus, the method presented here is to our knowledge the currently most sensitive assay available for plasma estrogen measurements in the ultra-low range and, as such, a reliable tool for a proper evaluation of potent aromatase inhibitors and other potential drugs influencing on plasma estrogen levels.

Treatment with high-dose estrogen (diethylstilbestrol) significantly decreases plasma estrogen and androgen levels but does not influence in vivo aromatization in postmenopausal breast cancer patients.

While growth factors and hormones are known to influence aromatase expression in experimental systems, little is know about potential factors influencing peripheral aromatization in postmenopausal women. The fact that peripheral aromatase activity is higher in old compared to young women and the finding of relatively high tissue estradiol (E2) concentrations after the menopause suggests peripheral aromatization could be influenced by estrogen concentration. To test this hypothesis, we determined plasma hormone levels (n=9) and in vivo aromatization (n=3) in postmenopausal women suffering from advanced breast cancer before and during treatment (4 weeks) with diethylstilbestrol (DES) 5mg three times daily. Plasma levels of cortisol (C), corticosteroid-binding globulin (CBG), and sex hormone binding globulin (SHBG) were significantly increased in all patients (P<0.05 for all). While we found no change in total body aromatization and plasma estrone (E(1)) levels, estradiol (E(2)) and estrone sulfate (E(1)S) were suppressed by a mean of 48.8 and 68.2%, respectively (P=0.043 and 0.008). Surprisingly, plasma levels of androstenedione (A), testosterone (T), dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) were also suppressed by a mean in the range of 32.1 to 52.6% (P<0.05 for all androgens). In contrast, no change in plasma progesterone or 17alpha-hydroxyprogesterone was found. Thus, one possible explanation to our findings could be that DES administered in high doses reduces 17,20-lyase activity in the adrenal gland.

Prognostic and predictive value of changes in tumour cell proliferation in locally advanced breast cancer primarily treated with doxorubicin.

We previously reported that high tumour cell proliferation evaluated by Ki-67 expression, high mitotic frequency and high histological grade were associated with resistance to primary doxorubicin monotherapy in locally advanced breast cancer harbouring wild-type (wt) TP53. The aim of our present study was to evaluate the predictive and prognostic impact of proliferation parameters assessed in tumour tissue obtained after chemotherapy, and alterations induced in tumour cell proliferation. While we found a significant reduction in Ki-67 expression and mitotic frequency in tumours with wtTP53 (p=0.001 and p=0.008, respectively), no significant change was recorded in tumours expressing mutant TP53. For histological grade there was no significant change in either group. There was a direct correlation between pre- and post-treatment values for Ki-67 and mitotic frequency in tumours harbouring wtTP53 (p=0.0001 for both), but no correlation in tumours harbouring mutated TP53. High post-treatment Ki-67 expression and mitotic frequency were found to predict doxorubicin resistance only in patients with wtTP53 (p=0.04 and p=0.03, respectively). The prognostic importance of proliferation markers and histological grade was found to be similar whether they were determined in the pre- or post-treatment samples (Ki-67; pre: p=0.02; post: p=0.03; mitotic frequency; p=0.002 and p=0.01, respectively; histological grade; p=0.0001 and p=0.002, respectively). While the reduction in mitotic frequency was associated with improved survival (p=0.03), no significant associations between changes in other parameters and outcome were recorded.