Associations between tamoxifen, estrogens, and FSH serum levels during steady state tamoxifen treatment of postmenopausal women with breast cancer.

BACKGROUND: The cytochrome P450 (CYP) enzymes 2C19, 2D6, and 3A5 are responsible for converting the selective estrogen receptor modulator (SERM), tamoxifen to its active metabolites 4-hydroxy-tamoxifen (4OHtam) and 4-hydroxy-N-demethyltamoxifen (4OHNDtam, endoxifen). Inter-individual variations of the activity of these enzymes due to polymorphisms may be predictors of outcome of breast cancer patients during tamoxifen treatment. Since tamoxifen and estrogens are both partly metabolized by these enzymes we hypothesize that a correlation between serum tamoxifen and estrogen levels exists, which in turn may interact with tamoxifen on treatment outcome. Here we examined relationships between the serum levels of tamoxifen, estrogens, follicle-stimulating hormone (FSH), and also determined the genotypes of CYP2C19, 2D6, 3A5, and SULT1A1 in 90 postmenopausal breast cancer patients. METHODS: Tamoxifen and its metabolites were measured by liquid chromatography-tandem mass spectrometry. Estrogen and FSH levels were determined using a sensitive radio- and chemiluminescent immunoassay, respectively. RESULTS: We observed significant correlations between the serum concentrations of tamoxifen, N-dedimethyltamoxifen, and tamoxifen-N-oxide and estrogens (p < 0.05). The genotype predicted CYP2C19 activity influenced the levels of both tamoxifen metabolites and E1. CONCLUSIONS: We have shown an association between tamoxifen and its metabolites and estrogen serum levels. An impact of CYP2C19 predicted activity on tamoxifen, as well as estrogen kinetics may partly explain the observed association between tamoxifen and its metabolites and estrogen serum levels. Since the role of estrogen levels during tamoxifen therapy is still a matter of debate further prospective studies to examine the effect of tamoxifen and estrogen kinetics on treatment outcome are warranted.

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.

Letrozole is superior to anastrozole in suppressing breast cancer tissue and plasma estrogen levels.

PURPOSE: To evaluate the influence of the third-generation aromatase inhibitor letrozole (Femara) on breast cancer tissue levels of estrone (E(1)), estradiol (E(2)), and estrone sulfate (E(1)S) in postmenopausal women undergoing primary treatment for locally advanced estrogen receptor/progesterone receptor-positive breast cancers. EXPERIMENTAL DESIGN: Breast cancer tissue samples were collected before and following 4 months of neoadjuvant therapy with letrozole (2.5 mg o.d.), and tissue estrogen levels measured using a highly sensitive RIA after high-pressure liquid chromatography purification. RESULTS: Letrozole suppressed pretreatment tumor levels of E(2), E(1), and E(1)S by 97.6%, 90.7%, and 90.1%, respectively. These data reveal that letrozole suppresses tissue estrogen levels significantly below what has previously been recorded with anastrozole (89.0%, 83.4%, and 72.9% suppression, respectively) using the same methods. To confirm the differential effect of letrozole and anastrozole on each plasma estrogen fraction, we re-analyzed plasma samples obtained from a previous intrapatient cross-over study comparing letrozole and anastrozole using an improved RIA (detection limits of 0.67, 1.14, and 0.55 pmol/L for E(2), E(1), and E(1)S, respectively). Letrozole consistently suppressed each plasma estrogen fraction below the levels recorded for anastrozole: E(2) (average suppression by 95.2% versus 92.8%; P = 0.018), E(1) (98.8% suppression versus 96.3%; P = 0.003), and E(1)S (98.9% suppression versus 95.3%; P = 0.003). CONCLUSION: Our data reveals that letrozole (2.5 mg o.d.) is more effective compared with anastrozole (1.0 mg o.d.) with respect to tissue as well as plasma estrogen suppression in patients with postmenopausal breast cancer.

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.

Total body aromatization in postmenopausal breast cancer patients is strongly correlated to plasma leptin levels.

The adipocytokine leptin has recently been shown to enhance the expression of aromatase via promoter II and I.3 using an AP-1 motif. Thus, we evaluated the correlation between plasma leptin concentrations and total body aromatization (TBA) as well as plasma levels of estrone (E(1)), estradiol (E(2)) and estrone sulfate (E(1)S) in postmenopausal breast cancer patients. Twenty-two postmenopausal women with metastatic breast cancer, participating in tracer studies for the measurement of total body aromatization (TBA) in vivo, were available. In addition, blood samples for plasma estrogens and leptin measurements were available from another 22 breast cancer patients and 114 healthy postmenopausal women participating in the mammography-screening program. Values for TBA varied from 1.46 to 4.72% while plasma leptin levels ranged from 1.83 to 95.51 ng/ml in the same group of patients. All plasma estrogen levels were in the normal range expected for postmenopausal women. We found a significant correlation between pretreatment leptin levels and TBA (r(s) 0.452, P=0.01). In contrast, basal levels of TBA did not correlate to body mass index (BMI) in the same group of patients. Plasma leptin levels correlated to plasma levels of estradiol (r(s) 0.659, P=0.007), and estrone sulfate (r(s) 0.562, P=0.01) in the group of breast cancer patients (n=44) as well as in the group of healthy postmenopausal women (estradiol, r(s) 0.363, P< or =0.001, estrone sulfate r(s) 0.353, P< or =0.001). In conclusion, we found plasma leptin levels to correlate to TBA in breast cancer patients and to plasma levels of estradiol and estrone sulfate in breast cancer patients as well as in healthy postmenopausal females. These findings suggest that leptin may influence on aromatase activity in vivo, providing a possible link between body weight and plasma estrogen levels as well as breast cancer risk.

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.

The IGF-system in healthy pre- and postmenopausal women: relations to demographic variables and sex-steroids.

Plasma insulin-like growth factor (IGF)-I, free IGF-I and -II, IGF-binding protein (IGFBP)-1, -2, and -3 together with IGFBP-3 protease activity were measured in 114 postmenopausal and 39 premenopausal healthy women. For each parameter, the mathematical distribution was characterised, and the normal range for pre- and postmenopausal women described, together with correlations to demographic variables and sex-steroids (postmenopausal women). Postmenopausal women had lower levels of plasma IGF-I (P<0.001) and free IGF-I (P<0.001) compared to premenopausal women, while plasma IGFBP-2 (P<0.05) and immunoreactive IGFBP-3 (P<0.001) were higher in postmenopausal women. Free IGF-I (but none of the other parameters) was significantly lower in postmenopausal smokers compared to non-smokers (P<0.05).IGF-I and -II both correlated positively to height (r=0.203, P<0.05 and r=0.198, P<0.05, respectively), while IGF-II correlated positively to weight (r=0.250, P<0.01). Plasma IGF-I correlated positively to androstenedione (r=0.292, P<0.01) and dehydroepiandrosterone sulphate (DHEAS, r=0.202, P<0.05), while a significant positive correlation was observed between IGF-II on the one side and oestradiol (E(2), r=0.227), oestrone sulphate (E(1)S, r=0.238) and androstenedione (r=0.213) on the other side (P<0.05 for all). Our results support a relation between sex-steroids and IGF-I and -II in healthy postmenopausal women. The lower levels of total and free IGF-I in postmenopausal compared to premenopausal women indicate lower bioavailability of this growth factor in elderly females.