Dagfinn Ekse

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 (E1), estradiol (E2), and estrone sulfate (E1S) 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 E2, E1, and E1S 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 E2, E1, and E1S, respectively). Letrozole consistently suppressed each plasma estrogen fraction below the levels recorded for anastrozole: E2 (average suppression by 95.2% versus 92.8%; P = 0.018), E1 (98.8% suppression versus 96.3%; P = 0.003), and E1S (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.

Tissue estradiol is selectively elevated in receptor positive breast cancers while tumour estrone is reduced independent of receptor status

Previous studies have suggested elevated estrogen production in tumour-bearing breast quadrants as well as in breast cancers versus benign tissue. Using highly sensitive assays, we determined breast cancer tissue estrogen concentrations together with plasma and benign tissue estrogen concentrations in each quadrant obtained from mastectomy specimens (34 postmenopausal and 13 premenopausal women). We detected similar concentrations of each of the three major estrogens estradiol (E(2)), estrone (E(1)) and E(1)S in tumour-bearing versus non-tumour-bearing quadrants. Considering malignant tumours, intratumour E(1) levels were reduced in cancer tissue obtained from pre- as well as postmenopausal women independent of tumour ER status (average ratio E(1) cancer: benign tissue of 0.2 and 0.3, respectively; p<0.001 for both groups), suggesting intratumour aromatization to be of minor importance. The most striking finding was a significant (4.1-8.6-fold) increased E(2) concentration in ER positive tumours versus normal tissue (p<0.05 and <0.001 for pre- and postmenopausal patients, respectively), contrasting low E(2) concentrations in ER- tumours (p<0.01 and <0.001 comparing E(2) levels between ER+ and ER- tumours in pre- and postmenopausals, respectively). A possible explanation to our finding is increased ligand receptor binding capacity for E(2) in receptor positive tumours but alternative factors influencing intratumour estrogen disposition cannot be excluded.

Relations between sex hormones, sex hormone binding globulin, insulin-like growth factor-I and insulin-like growth factor binding protein-1 in post-menopausal breast cancer patients.

OBJECTIVE Oestrogens, androgens and anti‐endocrine drugs such as tamoxifen and aminoglutethimide influence plasma Insulin‐like growth factor‐I (IGF‐I). IGF‐I, in turn, has been found to stimulate the peripheral aromatase in vitro. The aim of this study was to examine relations between sex hormones, IGF‐I and insulin‐like growth factor binding protein‐1 (IGFBP‐1) In post‐menopausal women with breast cancer.

Influence of tamoxifen on sex hormones, gonadotrophins and sex hormone binding globulin in postmenopausal breast cancer patients.

Estrone sulphate (E1S) may be an important estrogen source in breast cancers, particularly in postmenopausal women. Recent studies have shown that tamoxifen inhibits the uptake and metabolism of E1S to estradiol (E2) in cell cultures. To evaluate a possible influence of tamoxifen on E1S disposition in vivo, we measured plasma levels of E1S together with unconjugated estrogens (E1 and E2), androgens (T, A, DHEA and DHEAS), SHBG, FSH and LH in 32 postmenopausal breast cancer patients before and during tamoxifen treatment. In a subgroup of 10 patients, we measured 24 h urinary excretion of estrogen metabolites to evaluate the influence of tamoxifen treatment on estrogen metabolism and total estrogen production. Tamoxifen increased plasma levels of E1S (mean increase of 18.1%, P < 0.05) and the ratio of E1S/E1 (mean increase of 25.7%, P < 0.01) and E1S/E2 (mean increase of 34.7%, P < 0.0005). No significant change in plasma E1 was seen, but plasma E2 was reduced (mean reduction of 12.1%, P < 0.005). The fall in plasma E2 was probably secondary to a fall in plasma T (mean reduction of 11.9%, P < 0.05) due to a reduced ovarian excretion of this androgen. The mechanisms may be a reduced gonadotrophin stimulation of the ovary, as plasma FSH and LH fell by mean values of 45.5 and 48.1%, respectively (P < 0.0001 for both). The increase in plasma E1S was accompanied by a reduced ratio of 2OHE1/E1 in urine (mean reduction of 38.2%, P < 0.025) indicating reduced 2-hydroxylation. Possible mechanisms for these alterations are discussed.

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.

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

BackgroundThe 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.MethodsTamoxifen 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.ResultsWe 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.ConclusionsWe 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.

A sensitive assay for measurement of plasma estrone sulphate in patients on treatment with aromatase inhibitors

A major obstacle to the understanding of the mechanisms of action of aromatase inhibitors in breast cancer is the observation that plasma estrogens are sustained at about 30-50% of their control levels despite 85-95% inhibition of the conversion of tracer androstenedione (A) to estrone (E1). The discrepancy could be due to lack of sensitivity of current RIAs. Due to low levels of plasma estradiol (E2) (mean about 20 pM) and E1 (mean about 75 pM) in postmenopausal women, it is difficult to develop RIA methods with the sensitivity required to detect > 90% suppression from baseline. In contrast, the plasma level of the estrogen conjugate estrone sulphate (E1S) is substantially higher (mean level about 400 pM). This paper describes a new assay to measure plasma E1S in the low range aiming to detect > 95% suppression of E1S from baseline values in patients treated with aromatase inhibitors. E1S was separated from unconjugated estrogens, hydrolysed and purified as unconjugated E1. E1 was subsequently reduced to E2, purified, and measured by a highly sensitive RIA using oestradiol-6-(O-carboxymethyl) oximino-(2(-)[125I]iodohistamine as ligand. The sensitivity limit of the method was 2.7 pM. Patients on treatment with the aromatase inhibitors formestane or aminoglutethimide or both drugs in concert were found to have plasma levels of E1S ranging from 3 to 274 pM with a mean suppression of 78, 86 and 95%, respectively, compared to baseline, a lower suppression than that reported in previous trials with these drugs.

Influence of plasma estrogen levels on the length of the disease-free interval in postmenopausal women with breast cancer

SummaryThe influence of plasma estrogen levels on disease-free interval (time from primary treatment to first relapse, DFI) in breast cancer patients is not known. Any relation between plasma estrogens and the outcome in breast cancer patients may have implications considering use of hormone replacement therapy (HRT) in patients treated for breast cancer. We measured plasma estradiol (E2), estrone (E1), and estrone sulfate (E1S) in 92 postmenopausal women with breast cancer relapse and correlated plasma estrogen levels to the length of their disease-free interval (DFI1) and the length of the DFI in the subgroup of patients in whom this extended a time period of more than 2 years (DFI2). The length of DFI2 correlated negatively to plasma level of E1S (p < 0.025) and E2 (p < 0.05) and to the E2/E1 and E1S/E1 ratios (p < 0.05), while the length of DFI1 correlated negatively to plasma level of E1S (p < 0.025) and the E1S/E1 ratio (p < 0.005). We also analyzed for possible correlations between DFIs and plasma estrogen levels in subgroups based on tumor stage at diagnosis and previous therapy. In general, these subgroup analyses revealed negative correlations of statistical significance or borderline significance between the DFI1 and DFI2 and E2 and the E2/E1 ratio and non-significant negative correlations between plasma levels of E1S and DFI1 and DFI2. In particular, strong negative correlations between plasma estrogen levels and the length of DFI1 and DFI2 were found among patients responding to first line endocrine treatment for relapse and among patients with primar stage III tumors. Our findings suggest plasma E2 and E1S to stimulate the growth of micrometastases in patients treated for breast cancer.

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.

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.