Molecular clustering of endometrial carcinoma based on estrogen-induced gene expression.

Identification of biomarkers potentially provides prognostic information that can help guide clinical decision-making. Given the relationship between estrogen exposure and endometrial cancer, especially low grade endometrioid carcinoma, we hypothesized that high expression of genes induced by estrogen would identify low risk endometrioid endometrial cancers. cDNA microarray and qRT-PCR verification were used to identify six genes that are highly induced by estrogen in the endometrium. These estrogen-induced biomarkers were quantified in 72 endometrial carcinomas by qRT-PCR. Unsupervised cluster analysis was performed, with expression data correlated to tumor characteristics. Time to recurrence by cluster was analyzed using the Kaplan-Meier method. A receiver operating characteristic (ROC) curve was generated to determine the potential clinical utility of the biomarker panel to predict prognosis. Expression of all genes was higher in endometrioid carcinomas compared to non-endometrioid carcinomas. Unsupervised cluster analysis revealed two distinct groups based on gene expression. The high expression cluster was characterized by lower age, higher BMI, and low grade endometrioid histology. The low expression cluster had a recurrence rate 4.35 times higher than the high expression cluster. ROC analysis allowed for the prediction of stage and grade with a false negative rate of 4.8% based on level of gene expression in endometrioid tumors. We have therefore identified a panel of estrogen-induced genes that have potential utility in predicting endometrial cancer stage and recurrence risk. This proof-of-concept study demonstrates that biomarker analysis may play a role in clinical decision making for the therapy of women with endometrial cancer.

Coordinate regulation of the production and signaling of retinoic acid by estrogen in the human endometrium.

To determine whether estrogen regulates retinoic acid (RA) production and signaling in the human endometrium as it does in the rodent uterus, we investigated the effects of estrogens on the expression of RA-metabolizing enzymes, retinoid receptors, and biomarker genes in the post- and premenopausal human endometrium. Real-time quantitative PCR revealed that retinaldehyde dehydrogenase (RALDH) 2, a critical enzyme in RA biosynthesis, was induced 4-fold by estrogen replacement therapy with either Premarin or a mixture of estrone and equilin sulfates for 3 months. Estrogen replacement therapy also increased the expression of the RA receptor RAR alpha 1.9-fold. In parallel, there was a marked increase in the expression of two RA-regulated genes, cellular retinoic acid-binding protein II and tissue transglutaminase. In the premenopausal endometrium, the levels of RALDH1, RALDH2, RAR alpha, and cellular retinoic acid-binding protein II were increased in the estrogen-dominated proliferative phase, and the transcripts for the RA catabolic enzyme retinoic acid 4-hydroxylase (CYP26A1) and tissue transglutaminase were significantly increased in the secretory phase. Our results suggest that estrogen coordinately up-regulates RA production and signaling in the human endometrium. This coordinate mechanism may play a role in the antiproliferative effects that counterbalance the estrogen-induced endometrial proliferation.

Pharmacokinetics of 17 beta-dihydroequilin sulfate in normal postmenopausal women under steady state conditions.

OBJECTIVE: In the present study, the constant infusion of [3H]17 beta-dihydroequilin sulfate ([3H]17 beta-EqS) was used to estimate the metabolic clearance rate (MCR) of 17 beta-dihydroequilin sulfate (17 beta-EqS) and to measure the conversion of this estrogen to equilin sulfate (EqS), equilenin sulfate (EqnS), 17 beta-dihydroequilenin sulfate (17 beta-EqnS), equilin (Eq), equilenin (Eqn), 17 beta-dihydroequilin (17 beta-Eq), and 17 beta-dihydroequilenin (17 beta-Eqn) in normal postmenopausal women. METHODS: In seven healthy postmenopausal women, infusion of [3H]17 beta-EqS was started 30 minutes after a priming dose and continued at a constant rate of 10-20 microCi/hour, for 3-6 hours. Three blood samples were taken during and at the end of infusion. From the plasma, unconjugated and sulfate-conjugated estrogens, 17 beta-EqS, EqS, EqnS, 17 beta-EqnS, Eq, Eqn, 17 beta-Eq, and 17 beta-Eqn were isolated and purified by high performance liquid chromatography. The MCR of 17 beta-EqS and the conversion ratios and transfer constants (rho) for precursor (17 beta-EqS) to products were calculated. RESULTS: The mean MCR of 17 beta-EqS was calculated to be 797 +/- 90 L/day or 506 +/- 60 L/m2 per day. The mean conversion ratio (CRPRE-PROBB) was 2.4 +/- 0.4 for EqS, 0.3 +/- 0.04 for EqnS, 0.25 +/- 0.03 for 17 beta-EqnS, 0.09 +/- 0.02 for Eq, 0.03 +/- 0.01 for Eqn, 0.08 +/- 0.02 for 17 beta-Eq, and 0.03 +/- 0.01 for 17 beta-Eqn. In both the sulfate-conjugated and unconjugated forms, the most abundant metabolite formed was Eq. Based on the previously reported MCR of EqS (170 L/m2 per day) and 17 beta-Eq (1252 L/m2 per day), the transfer constants [rho]BB were calculated to be 0.8 +/- 0.10 and 0.20 +/- 0.03, respectively. The results indicate that a large portion of 17 beta-EqS is converted to EqS and the more potent estrogen 17 beta-Eq. The ratio of rho EqS-17 beta-EqS to rho 17 beta-EqS-EqS was calculated to be 0.8 +/- 0.1 and represents the extent of C-17-oxidation and reduction and indicates that substantial amounts of 17 beta-reduced metabolites will still be present in the blood although the oxidation reaction was somewhat greater. CONCLUSION: The data indicate that, compared with the classic estrogens, the in vivo metabolism of ring B unsaturated estrogens is complex. Thus, although the amount of 17 beta-EqS originally present in the conjugated equine estrogens is small, the pharmacokinetics and pharmacodynamics of EqS, 17 beta-EqS, and the extensive interconversions between these estrogens support the hypothesis that the major in vivo activity of the EqS present in conjugated equine estrogens is expressed through its metabolism to 17 beta-EqS and 17 beta-Eq. Furthermore, the increased estrogenic activity associated with this drug may in part be due to the formation of these 17 beta-reduced metabolites.

Low-dose esterified estrogens (0.3 mg/day): long-term and short-term effects on menopausal symptoms and quality of life in postmenopausal women.

OBJECTIVE: To study the impact of low-dose unopposed esterified estrogens on menopausal symptoms and quality of life. METHODS: In a long-term, 2-year, randomized, double-blind, placebo-controlled study, 204 postmenopausal women were treated with esterified estrogens 0.3 mg daily or placebo. Menopausal symptoms were assessed with a modified Kupperman index at baseline, 3, 6 and thereafter every 6 months. In a second 12-week, open-label, short-term pilot study, 25 postmenopausal women with moderate to severe vasomotor symptoms were treated with esterified estrogens 0.3 mg daily for 12 weeks. Vasomotor symptoms and quality of life were assessed using the Greene scale and Quality of Life Menopause Scale (QUALMS). RESULTS: In the long-term study, significant (p < 0.05) reductions in total symptom scores were observed at each time point with esterified estrogens compared with placebo. Somatic symptom scores (hot flushes, night sweats, vaginal dryness) decreased significantly (p < 0.01) in patients treated with esterified estrogens 0.3 mg compared to baseline and placebo. In the short-term, open-label pilot study, the incidence of vasomotor symptoms was significantly (p < 0.01) reduced with esterified estrogens 0.3 mg from week 4 until the study end. Significant (p < 0.05) improvements versus baseline were seen in the somatic and vasomotor/sleep domains and in the total quality-of-life score. CONCLUSIONS: Esterified estrogens 0.3 mg given daily provide adequate menopausal symptom relief and improved quality of life in postmenopausal women.

Effects of estrogen on susceptibility to oxidation of low-density and high-density lipoprotein in postmenopausal women.

OBJECTIVE: To investigate the effects of estrogen on the susceptibility to oxidation of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in postmenopausal women. METHODS: A total of 23 postmenopausal women were treated with 0.625 mg of conjugated equine estrogen daily for 3 months. Blood samples were obtained before and after therapy. Plasma levels of total cholesterol and triglyceride and the concentrations of cholesterol, triglyceride, phospholipid in LDL and HDL were determined enzymatically and the levels of apolipoprotein A-I, A-II in HDL and apolipoprotein B in LDL were measured by turbidimetric immunoassay. The isolated LDL and HDL were incubated at 37 degrees C for 24 h with CuSO4 5 mumol/l and the lipid peroxide concentration of LDL and HDL was measured. RESULTS: Estrogen significantly reduced the plasma level of total cholesterol and significantly increased the plasma level of triglyceride. The LDL concentrations of cholesterol, phospholipid and apolipoprotein B were significantly decreased following estrogen therapy. The triglyceride level of LDL did not change significantly. The HDL concentrations of cholesterol, triglyceride, phospholipid and apolipoprotein A-I and A-II were all significantly elevated after estrogen therapy. Estrogen significantly inhibited the peroxidation of LDL at 50-2000 micrograms of LDL protein (14.17 +/- 4.17-11.49 +/- 1.42 nmol/200 micrograms of LDL protein, P < 0.001) and of HDL (4.49 +/- 1.74-3.37 +/- 1.24 nmol/200 micrograms of HDL protein, P < 0.03) induced by their incubation in the presence of CuSO4. CONCLUSIONS: Estrogen inhibited the susceptibility of LDL and HDL to oxidative modification and favorably affected lipid metabolism by reducing the number of LDL particles and increasing the number of HDL particles in plasma that were resistant to oxidation.

Leuprolide acetate depot and hormonal add-back in endometriosis: a 12-month study. Lupron Add-Back Study Group.

OBJECTIVE: To evaluate the efficacy and safety of a GnRH agonist, leuprolide acetate depot, alone and in combination with three hormonal add-back regimens in the management of endometriosis-associated pelvic pain. METHODS: Two hundred and one patients were enrolled in this multicenter, randomized, double-blind, 1-year trial. All patients were given an intramuscular injection of leuprolide acetate depot 3.75 mg every 4 weeks. Patients were assigned to one of four treatment groups: Group A received placebos for progestin and estrogen, group B received norethindrone acetate 5 mg daily and placebo for estrogen, group C received norethindrone acetate 5 mg and conjugated equine estrogens 0.625 mg daily, and group D received norethindrone acetate 5 mg and conjugated equine estrogens 1.25 mg daily. Pelvic pain scores were assessed monthly, and bone density was measured after 24 and 52 weeks. RESULTS: By week 8, all four groups showed significant improvement in pelvic pain scores compared with baseline levels. A higher proportion of group D patients terminated the study prematurely due to a lack of improvement in symptoms. Group A experienced a 6.3 +/- 2.3% (P < or = .001) loss in bone density after 52 weeks of treatment, whereas bone density was preserved in all three add-back groups. CONCLUSION: The use of leuprolide acetate depot in combination with norethindrone acetate 5 mg alone, or with norethindrone acetate and conjugated equine estrogens 0.625 mg, provides effective suppression of pelvic pain symptoms associated with endometriosis while protecting against bone loss.

Biologic effects of 17 alpha-dihydroequilin sulfate.

OBJECTIVE: To determine the independent biologic effects of 17 alpha-dihydroequilin sulfate. DESIGN: Prospective randomized study. SETTING: University of Southern California Medical Center. PATIENTS(S): Twenty-one postmenopausal women, mean age 50 +/- 2 (+/-SEM) years, and mean body mass index 27 +/- 2. INTERVENTION(S): Women were randomized to receive daily oral doses of either 1.25 mg of estrone sulfate (E1S), 0.2 mg of 17 alpha-dihydroequilin sulfate, or a combination. Three blood and urine samples were obtained before and after 30 and 90 days of treatment. RESULT(S): After 30 and 90 days of treatment, E1S alone increased sex hormone-binding globulin (SHBG) levels significantly, 19.7% +/- 6.0% and 61.3% +/- 13.0%, whereas 17 alpha-dihydroequilin sulfate reduced SHBG levels, 20.8% +/- 68% and 12.4% +/- 7.5%, respectively. Nevertheless, the combination of E1S and 17 alpha-dihydroequilin sulfate significantly increased SHBG levels, 103% +/- 27.9% and 98.2% +/- 19.1%, compared with baseline at 30 and 90 days. Fewer changes were evident with corticosteroid-binding globulin (CBG). After 90 days of treatment, CBG levels significantly increased 30.9% +/- 5.5% with E1S, decreased by 7.2% +/- 5.0% with 17 alpha-dihydroequilin sulfate, and, with the combination, significantly increased by 10.5% +/- 2.4% compared with baseline. Changes in lipids and lipoproteins were more variable. However, high-density-lipoprotein cholesterol increased significantly with E1S at 30 and 90 days compared with baseline, 96.5% +/- 39% and 91.5% +/- 22.6%, and with the combination increased 66.4% +/- 13.3% and 79.2% +/- 24.4%, respectively. Fewer changes were evident with 17 alpha-dihydroequilin sulfate alone, decreasing 4.4% +/- 22% and 2.6% +/- 21.3%. Urinary ratios of bone collagen equivalents-creatinine and calcium-creatinine decreased in all three groups. However, the combination group resulted in a significantly greater percentage decrease in bone collagen equivalents-creatinine than with E1S alone. CONCLUSIONS(S): 17 alpha-Dihydroequilin sulfate could modify some of the first-pass effects of conjugated equine estrogens and act synergistically with other conjugated equine estrogens to reduce bone resorption.

Metabolism of [3H] 17 beta-dihydroequilin and [3H] 17 beta-dihydroequilin sulfate in normal postmenopausal women.

The metabolism of 17 beta-dihydroequilin and 17 beta-dihydroequilin sulfate was investigated after intravenous administration of [3H] 17 beta-dihydroequilin and [3H] 17 beta-dihydroequilin sulfate to postmenopausal women. Urine was collected for 3 days and 46.2 +/- 10.5% and 54.5 +/- 8.7% of the injected dose of [3H] 17 beta-dihydroequilin and [17 beta-3H]dihydroequilin sulfate was excreted in the urine respectively. The estrogens present in urine were extracted and fractionated into unconjugated, sulfate, and glucuronide conjugated forms. With both precursors, the major amount (63-64%) of metabolites were excreted in the urine conjugated with glucuronic acid. From the unconjugated, sulfate, and glucuronide fraction, 17 beta-dihydroequilenin, 17 beta-dihydroequilin, equilenin, and equilin were isolated. The conversions with both precursors were similar and 17 beta-dihydroequilenin was the major metabolite isolated from all three fractions; however, the highest levels of all four metabolites were present in the glucuronide fraction. Along with these identifiable metabolites, a large amount (51-81%) of radioactivity was present in the form of metabolites which are more polar than any of the known ring-B unsaturated estrogens. These appear to be polyhydroxy 17 beta-reduced ring-B unsaturated estrogens which remain to be identified. The in-vivo formation of equilenin and 17 beta-dihydroequilenin indicates the presence of the enzyme 6.8(9) steroid dehydrogenase in humans.

Metabolism of [3H]equilin-[35S]sulfate and [3H]equilin sulfate after oral and intravenous administration in normal postmenopausal women and men.

The absorption of equilin sulfate and equilin from the gastrointestinal tract was determined in normal men after the ingestion of [3H]equilin-[35S]sulfate or a mixture of [3H]equilin and equilin-[35S]sulfate, while the metabolism of equilin sulfate was investigated after iv administration of [3H]equilin sulfate to postmenopausal women. After the oral administration of [3H]equilin-[35S]sulfate, equilin sulfate containing both 3H and 35S was isolated from plasma; however, only in the first sample taken at 10 min was the 3H/35S ratio the same as that of the [3H]equilin-[35S]sulfate ingested. The 3H/35S ratio then increased, and by 12 h only traces of equilin-[35S]sulfate were detectable. Similarly, after the ingestion of [3H]equilin and equilin-[35S]sulfate, [3H]equilin-[35S]sulfate was isolated from plasma. The 3H/35S ratio was at all time points greater than the 3H/35S ratio of the ingested mixture. Analysis of urine indicated that over 98% of 35S was not associated with any steroid and was most likely inorganic sulfate. After iv administration of [3H] equilin sulfate to postmenopausal women, equilin, equilenin, 17 beta-dihydroequilin, and 17 beta-dihydroequilenin were isolated from the urine. These results indicate that 1) some of the orally administered equilin sulfate was absorbed from the gut without prior hydrolysis, 2) some equilin sulfate was hydrolyzed in the gut before absorption; 3) equilin was absorbed more efficiently than equilin sulfate; 4) equilin absorbed was readily sulfated and circulated in this form; and 5) equilin sulfate was extensively metabolized, and the metabolites were excreted in the urine mainly conjugated with glucuronic acid.

Biologic effects of equilin sulfate in postmenopausal women.

In order to determine the relative potency of equilin sulfate (EqS), a major constituent of conjugated equine estrogens, 15 women received oral doses of EqS (0.15, 0.31, and 0.625 mg) for 25 days. Doses of 0.31 and 0.625 mg significantly stimulated hepatic globulins. This stimulatory effect ranged from being 1.5 to 8 times greater than the effects of comparable doses of estrone sulfate and conjugated equine estrogens. A significant stimulation in high-density lipoprotein-cholesterol occurred with as little as 0.15 mg of EqS. Elevations in the high-density lipoprotein/low-density lipoprotein-cholesterol ratio occurred with EqS, which resulted in an approximately 4-fold greater response than that achieved with comparable doses of conjugated equine estrogens. The fasting urinary calcium/creatinine ratio was only significantly lowered with 0.625 mg of EqS and was less potent than conjugated equine estrogens in this regard. It is concluded that EqS is a potent estrogen that contributes significantly to the hepatic stimulatory effects of conjugated equine estrogens. These data also provide support for the suggestion that there may be a dissociation in potency between estrogenic effects on liver and bone.

The metabolism of equilin in normal men.

Healthy adult males received either [3H]-equilin intravenously (one subject) or a larger mass of unlabelled equilin orally (three subjects). Blood samples were taken at 20, 40, 60, 90 and 120 min and every h thereafter until eight h after injection. Urine was collected in 24 h aliquots for five days from all subjects. The half-life of the disappearance of the unconjugated radioactivity from blood was 30 min and that in the conjugated sulfate fraction was 5 1/2 h. Approximately 50% of the injected radioactivity was recovered in the urine over 5 days. After extraction, hydrolysis and fractionation, most (83%) of the radioactive material found in the urine was present in the glucuronide fraction while only 2 and 6% were present in the unconjugated and sulfate fractions, respectively. The three fractions were combined for further isolation and identification of the metabolites. Radiochemically pure equilin, equilenin, 17 beta-dihydro-equilin and 17 beta-dihydroequilenin were isolated and identified but the largest fraction of radioactivity (70.5%) was present in the form of metabolites which are more polar than any of the known ring B unsaturated estrogens. These appear to be polyhydroxy 17-reduced ring B unsaturated estrogens. These results indicate that the ring B unsaturated estrogen equilin is being metabolized in man in a somewhat similar manner to that of the classical estrogen estrone. Knowledge of the formation of 17 beta-dihydroequilin from equilin in man is of importance because this estrogen is approximately 8 times more potent as a uterotrophic agent than the commonly used estrogen, equilin.

[Equilin sulphate in the treatment of the menopausal syndrome (author's transl)].

0.25 sodium equilin sulphate and 0.625 mg Premarin were compared as to their effects in 12 postmenopausal syndrome. The initial daily dosage of 0.25 mg equilin had a stronger oestrongenic effect in the alleviation of vasomotor disturbances than subsequently-administered Premarin. No significant difference was found in effect on the vaginal epithelium or the endometrium or in the incidence of irregular bleeding. The number of women in whom withdrawal bleeding was reported was considerably reduced by lowering the dosage to 0.2 mg equilin sulphate. Consequently 0.2 to 0.3 mg equilin sulphate must be considered the optimum dosage.