Prognostic impact of genetic variants of CYP19A1 and UGT2B17 in a randomized trial for endocrine-responsive postmenopausal breast cancer.

Polymorphisms of genes involved in estrogen synthesis have been linked to breast cancer risk, prognosis, and treatment response. We investigated the prognostic impact of a deletion spanning the entire UGT2B17 gene (UGT2B17*2) and genetic variants of the aromatase CYP19A1 and estrogen receptor α (ESR1) in 125 postmenopausal women with ER-positive breast cancer enrolled in a randomized pre-surgical trial. The UGT2B17*2 was estimated by copy number variation assays and the CYP19A1 rs10046/rs4646 and ESR1 rs2077647/rs2234693/rs9340799 by TaqMan allelic discrimination assays. Serum exemestane/17-hydroxy exemestane were determined by MS and estrone (E1)/estradiol (E2)/ by GC-MS/MS. The association of genetic polymorphisms with "any event" was assessed by the Cox proportional hazards models adjusted for confounders. The UGT2B17*2 was associated with higher levels of 17-hydroxy exemestane (P = 0.04) and better prognosis (HR = 0.45; 95% CI: 0.20-1.01; P = 0.05) compared with homozygote UGT2B17 wt. The CYP19A1 rs10046 A and rs4646 C alleles were associated with higher estrogen levels: rs10046 AA vs. AG/GG genotypes had median E1 of 35.9 vs. 27.4 pg/mL (P = 0.05) and E2 of 7.57 vs. 3.9 pg/mL (P < 0.004). After a median follow-up of 7 years, women carrying the "low estrogen" alleles rs10046 G and rs4646 A had a better prognosis compared with homozygote wt for both polymorphisms (HR = 0.40; 95% CI: 0.17-0.93; P = 0.03). Our analysis points to an impact of UGT2B17 and CYP19A1 in postmenopausal endocrine responsive breast cancer. Carriers of UGT2B17*2 and CYP19A1 low estrogen variants may have better prognosis, supporting studies addressing the role of these polymorphisms in optimizing endocrine therapy. Trial registration: http://www.isrctn.com/ISRCTN86894592.

Drug monitoring of tamoxifen metabolites predicts vaginal dryness and verifies a low discontinuation rate from the Norwegian Prescription Database.

PURPOSE: Tamoxifen is an important targeted endocrine therapy in breast cancer. However, side effects and early discontinuation of tamoxifen remains a barrier for obtaining the improved outcome benefits of long-term tamoxifen treatment. Biomarkers predictive of tamoxifen side effects remain unidentified. The objective of this prospective population-based study was to investigate the value of tamoxifen metabolite concentrations as biomarkers for side effects. A second objective was to assess the validity of discontinuation rates obtained through pharmacy records with the use of tamoxifen drug monitoring. METHODS: Longitudinal serum samples, patient-reported outcome measures and pharmacy records from 220 breast cancer patients were obtained over a 6-year period. Serum concentrations of tamoxifen metabolites were measured by LC-MS/MS. Associations between metabolite concentrations and side effects were analyzed by logistic regression and cross table analyses. To determine the validity of pharmacy records we compared longitudinal tamoxifen concentrations to discontinuation rates obtained through the Norwegian Prescription database (NorPD). Multivariable Cox regression models were performed to identify predictors of discontinuation. RESULTS: At the 2nd year of follow-up, a significant association between vaginal dryness and high concentrations of tamoxifen, Z-4'-OHtam and tam-NoX was identified. NorPD showed a tamoxifen-discontinuation rate of 17.9% at 5 years and drug monitoring demonstrated similar rates. Nausea, vaginal dryness and chemotherapy-naive status were significant risk factors for tamoxifen discontinuation. CONCLUSIONS: This real-world data study suggests that measurements of tamoxifen metabolite concentrations may be predictive of vaginal dryness in breast cancer patients and verifies NorPD as a reliable source of adherence data.

A pooled analysis of CYP2D6 genotype in breast cancer prevention trials of low-dose tamoxifen.

Decreased CYP2D6 activity is associated with lower levels of active tamoxifen metabolites. We examined the impact of CYP2D6 genotype on tamoxifen pharmacokinetics, biomarker activity, and efficacy in a pooled analysis of low-dose tamoxifen. Four randomized breast cancer prevention trials of very-low-dose (1 mg/day, n = 52 or 10 mg/week, n = 152) or low-dose tamoxifen (5 mg/day, n = 171) were pooled. DNA from 367 subjects was genotyped for CYP2D6 alleles associated with absent (PM allele: *3, *4, *5, *6, *7, *8, *12, and *14), reduced (IM allele: *9, *10, *17, *29, *41), normal (EM allele), or increased (UM: *XN) enzyme activity. Associations of tamoxifen, metabolites, activity biomarkers, and event-free survival with rapid (UM/EM, UM/IM, EM/EM, EM/IM, or EM/PM alleles) versus slow metabolizers (PM/IM or PM/PM) were investigated through random effects models, with 'study' as the random factor, and Cox regression models, adjusting for confounders. Rapid metabolizers had higher endoxifen levels than slow metabolizers: 15.3 versus 12.2 ng/mL (P = 0.018) with 5 mg/day, and 3.8 versus 2.8 ng/mL (P = 0.004) with 1 mg/day or 10 mg/week tamoxifen. The IGF-I decrease correlated with endoxifen (P = 0.002) and 4-hydroxytamoxifen levels, demonstrating steeper decreases at higher metabolite levels (P = 0.001). After a median follow-up of 12 years, rapid metabolizers with prior history of breast neoplasms allocated to tamoxifen 5 mg/day had a 60 % reduction of risk of recurrences (HR = 0.40, 95 % CI: 0.16-0.99) compared to slow metabolizers. CYP2D6 genotype may have an impact on tamoxifen efficacy at low doses. Trials investigating tamoxifen dose adjustments based on the woman's hormonal context and CYP2D6 genotype are warranted.

Phospholipids from herring roe improve plasma lipids and glucose tolerance in healthy, young adults.

BACKGROUND: Herring roe is an underutilized source of n-3 polyunsaturated fatty acids (PUFAs) for human consumption with high phospholipid (PL) content. Studies have shown that PL may improve bioavailability of n-3 PUFAs. Arctic Nutrition's herring roe product MOPL™30 is a PL: docosahexaenoic acid (DHA)-rich fish oil mixture, with a DHA:eicosapentaenoic acid (EPA) ratio of about 3:1, which is also rich in choline. In this pilot study, we determined if MOPL30 could favorably affect plasma lipid parameters and glucose tolerance in healthy young adults. METHODS: Twenty female and one male adults, between 22 and 26 years of age, participated in the study. Participants took encapsulated MOPL30, 2.4 g/d EPA + DHA, for 14 days, and completed a three-day weighed food record before and during the capsule intake. Plasma lipids and their fatty acid (FA) composition, plasma and red blood cell (RBC) phosphatidylcholine (PC) FA composition, acylcarnitines, choline, betaine and insulin were measured before and after supplementation (n = 21), and one and four weeks after discontinuation of supplementation (n = 14). An oral glucose tolerance test was performed before and after supplementation. RESULTS: Fasting plasma triacylglycerol and non-esterified fatty acids decreased and HDL-cholesterol increased after 14 days of MOPL30 intake (p < 0.05). The dietary records showed that PUFA intake prior to and during capsule intake was not different. Fasting plasma glucose was unchanged from before to after supplementation. However, during oral glucose tolerance testing, blood glucose at both 10 and 120 min was significantly lower after supplementation with MOPL30 compared to baseline measurements. Plasma free choline and betaine were increased, and the n-6/n-3 polyunsaturated (PUFA) ratio in plasma and RBC PC were decreased post-supplementation. Four weeks after discontinuation of MOPL30, most parameters had returned to baseline, but a delayed effect was observed on n-6 PUFAs. CONCLUSIONS: Herring roe rich in PL improved the plasma lipid profile and glycemic control in young adults with an overall healthy lifestyle.

A randomized phase II presurgical trial of weekly low-dose tamoxifen versus raloxifene versus placebo in premenopausal women with estrogen receptor-positive breast cancer.

INTRODUCTION: We previously demonstrated that 1 or 5 mg per day of tamoxifen (T) given for four weeks before surgery reduces Ki-67 in breast cancer (BC) patients to the same extent as the standard 20 mg/d. Given the long half-life of T, a weekly dose (10 mg per week (w)) may be worth testing. Also, raloxifene (R) has shown Ki-67 reduction in postmenopausal patients in a preoperative setting, but data in premenopausal women are limited. We conducted a randomized trial testing T 10 mg/w vs. R 60 mg/d vs. placebo in a presurgical model. METHODS: Out of 204 screened subjects, 57 were not eligible, 22 refused to participate and 125 were included in the study. The participants were all premenopausal women with estrogen receptor-positive BC. They were randomly assigned to either T 10mg/w or R 60 mg/d or placebo for six weeks before surgery. The primary endpoint was tissue change of Ki-67. Secondary endpoints were modulation of estrogen and progesterone receptors and several other circulating biomarkers. RESULTS: Ki-67 was not significantly modulated by either treatment. In contrast, both selective estrogen receptor modulators (SERMs) significantly modulated circulating IGF-I/IGFBP-3 ratio, cholesterol, fibrinogen and antithrombin III. Estradiol was increased with both SERMs. Within the tamoxifen arm, CYP2D6 polymorphism analysis showed a higher concentration of N-desTamoxifen, one of the tamoxifen metabolites, in subjects with reduced CYP2D6 activity. Moreover, a reduction of Ki-67 and a marked increase of sex hormone-binding globulin (SHBG) were observed in the active phenotype. CONCLUSIONS: A weekly dose of tamoxifen and a standard dose of raloxifene did not inhibit tumor cell proliferation, measured as Ki-67 expression, in premenopausal BC patients. However, in the tamoxifen arm women with an extensive phenotype for CYP2D6 reached a significant Ki-67 modulation.

Serum concentrations of tamoxifen and its metabolites increase with age during steady-state treatment.

It has been suggested that the concentrations of tamoxifen and its demethylated metabolites increase with age. We measured the serum concentrations of the active tamoxifen metabolites, 4OHtamoxifen (4OHtam), 4-hydroxy-N-desmethyltamoxifen (4OHNDtam, Endoxifen), tamoxifen and its demethylated metabolites. Their relations to age were examined. One hundred fifty-one estrogen receptor and/or progesterone receptor positive breast cancer patients were included. Their median (range) age was 57 (32-85) years. Due to the long half-life of tamoxifen, only patients treated with tamoxifen for at least 80 days were included in the study in order to insure that the patients had reached steady-state drug levels. Tamoxifen and its metabolites were measured by liquid chromatography-tandem mass spectrometry. Their serum concentrations were related to the age of the patients. To circumvent effects of cytochrome (CYP) 2D6 polymorphisms we also examined these correlations exclusively in homozygous extensive metabolizers. The concentrations of 4OHNDtam, tamoxifen, NDtam (N-desmethyltamoxifen), and NDDtam (N-desdimethyltamoxifen) were positively correlated to age (n = 151, p = 0.017, 0.045, 0.011, and 0.001 respectively). When exclusively studying the CYP2D6 homozygous extensive metabolizers (n = 86) the correlation between 4OHNDtam and age increased (p = 0.008). Up to tenfold inter-patient variation in the serum concentrations was observed. The median (inter-patient range) concentration of 4OHNDtam in the age groups 30-49, 50-69, and >69 years were 65 (24-89), 116 (25-141), and 159 (26-185) ng/ml, respectively. We conclude that the serum concentrations of 4OHNDtam (endoxifen), tamoxifen, and its demethylated metabolites increase with age during steady-state tamoxifen treatment. This may represent an additional explanation why studies on the effects of CYP2D6 polymorphisms on outcome in tamoxifen-treated breast cancer patients have been inconsistent. The observed high inter-patient range in serum concentrations of tamoxifen and its metabolites, especially in the highest age group, suggest that use of therapeutic monitoring of tamoxifen and its metabolites is warranted.

Tissue distribution of 4-hydroxy-N-desmethyltamoxifen and tamoxifen-N-oxide.

Tamoxifen dosage is based on the one-dose-fits-all approach. The anticancer effect of tamoxifen is believed to be due to the metabolites, 4-hydroxytamoxifen (4OHtam), and 4-hydroxy-N-desmethyltamoxifen (4OHNDtam/endoxifen). These demethylated metabolites of tamoxifen have been associated with its side effects, whereas the effect mediated by tamoxifen-N-oxide (tamNox) is still poorly understood. Our objective was to improve the therapeutic index of tamoxifen by personalizing its dosage and maintaining serum tamoxifen metabolite concentrations within a target range. We examined the levels of tamoxifen, 4OHtam, 4OHNDtam, N-desmethyltamoxifen (NDtam), N-desdimethyltamoxifen (NDDtam), and tamNox in serum and in breast tumors specimens of 115 patients treated with 1, 5 or 20 mg/day of tamoxifen for 4 weeks before surgery in a randomized trial. Furthermore, the metabolism of tamNox in MCF-7 breast cancer cells was also studied. The concentrations of tamoxifen and its metabolites in tumor tissues were significantly correlated to their serum levels. Tumor tissue levels were 5-10 times higher than those measured in serum, with the exception of tamNox. In MCF-7 cells, tamNox was converted back to tamoxifen. In contrast to the tissue distribution of tamNox, the concentrations of 4OHtam and 4OHNDtam in tumor tissues corresponded to their serum levels. The results suggest that implementation of therapeutic drug monitoring may improve the therapeutic index of tamoxifen. Furthermore, the tissue distribution of tamNox deviated from that of the other tamoxifen metabolites.

Steroid receptor coactivators, HER-2 and HER-3 expression is stimulated by tamoxifen treatment in DMBA-induced breast cancer.

BACKGROUND: Steroid receptor coactivators (SRCs) may modulate estrogen receptor (ER) activity and the response to endocrine treatment in breast cancer, in part through interaction with growth factor receptor signaling pathways. In the present study the effects of tamoxifen treatment on the expression of SRCs and human epidermal growth factor receptors (HERs) were examined in an animal model of ER positive breast cancer. METHODS: Sprague-Dawley rats with DMBA-induced breast cancer were randomized to 14 days of oral tamoxifen 40 mg/kg bodyweight/day or vehicle only (controls). Tumors were measured throughout the study period. Blood samples and tumor tissue were collected at sacrifice and tamoxifen and its main metabolites were quantified using LC-MS/MS. The gene expression in tumor of SRC-1, SRC-2/transcription intermediary factor-2 (TIF-2), SRC-3/amplified in breast cancer 1 (AIB1), ER, HER-1, -2, -3 and HER-4, as well as the transcription factor Ets-2, was measured by real-time RT-PCR. Protein levels were further assessed by Western blotting. RESULTS: Tamoxifen and its main metabolites were detected at high concentrations in serum and accumulated in tumor tissue in up to tenfolds the concentration in serum. Mean tumor volume/rat decreased in the tamoxifen treated group, but continued to increase in controls. The mRNA expression levels of SRC-1 (P = 0.035), SRC-2/TIF-2 (P = 0.002), HER-2 (P = 0.035) and HER-3 (P = 0.006) were significantly higher in tamoxifen treated tumors compared to controls, and the results were confirmed at the protein level using Western blotting. SRC-3/AIB1 protein was also higher in tamoxifen treated tumors. SRC-1 and SRC-2/TIF-2 mRNA levels were positively correlated with each other and with HER-2 (P ≤ 0.001), and the HER-2 mRNA expression correlated with the levels of the other three HER family members (P < 0.05). Furthermore, SRC-3/AIB1 and HER-4 were positively correlated with each other and Ets-2 (P < 0.001). CONCLUSIONS: The expression of SRCs and HER-2 and -3 is stimulated by tamoxifen treatment in DMBA-induced breast cancer. Stimulation and positive correlation of coactivators and HERs may represent an early response to endocrine treatment. The role of SRCs and HER-2 and -3 should be further studied in order to evaluate their effects on response to long-term tamoxifen treatment.

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.

Validation and Determination of 25(OH) Vitamin D and 3-Epi25(OH)D3 in Breastmilk and Maternal- and Infant Plasma during Breastfeeding.

Vitamin D deficiency in pregnant women and their offspring may result in unfavorable health outcomes for both mother and infant. A 25hydroxyvitamin D (25(OH)D) level of at least 75 nmol/L is recommended by the Endocrine Society. Validated, automated sample preparation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were used to determine the vitamin D metabolites status in mother-infant pairs. Detection of 3-Epi25(OH)D3 prevented overestimation of 25(OH)D3 and misclassification of vitamin D status. Sixty-three percent of maternal 25(OH)D plasma levels were less than the recommended level of 25(OH)D at 3 months. Additionally, breastmilk levels of 25(OH)D decreased from 60.1 nmol/L to 50.0 nmol/L between six weeks and three months (p < 0.01). Furthermore, there was a positive correlation between mother and infant plasma levels (p < 0.01, r = 0.56) at 3 months. Accordingly, 31% of the infants were categorized as vitamin D deficient (25(OH)D < 50 nmol/L) compared to 25% if 3-Epi25(OH)D3 was not distinguished from 25(OH)D3. This study highlights the importance of accurate quantification of 25(OH)D. Monitoring vitamin D metabolites in infant, maternal plasma, and breastmilk may be needed to ensure adequate levels in both mother and infant in the first 6 months of infant life.

Induction of hepatic drug-metabolising enzymes and tamoxifen metabolite profile in relation to administration route during low-dose treatment in nude rats.

Tamoxifen is the most used anticancer drug and is approved for chemoprevention. Little is known about the enzyme inducing properties of low-dose regimens and the influence of route of administration. In this study, nude rats received 5 mg/kg/day of tamoxifen orally or a 50 mg continuous-release pellet subcutaneously. The mRNAs for cytochrome P450-enzymes (CYPs), flavin-containing monooxygenase 1 (FMO1) and phase II drug-metabolising enzymes were quantified by real-time RT-PCR. Tamoxifen and metabolite concentrations were measured using HPLC. We observed a significant increase in CYP3A18 and FMO1 mRNA expression levels in the orally treated animals, whereas the increase in CYP3A2 expression did not reach statistical significance (p=0.057). No significant induction of enzyme expression was observed in rats that received subcutaneous (S.c.) treatment. After 33 days the serum levels of 4-hydroxytamoxifen (4OHtam), tamoxifen and N-desmethyltamoxifen (NDtam) in orally treated animals were 1.8+/-0.7, 11.1+/-3.2 and 11.4+/-3.8 ng/ml, respectively. In subcutaneously treated animals, tamoxifen and N-desmethyltamoxifen were detected in tissues, but not in serum. These data demonstrate that in contrast to the subcutaneous administration, low-dose oral tamoxifen induced tamoxifen-metabolising enzymes. Furthermore, the different routes of administration resulted in different serum and tissue levels of tamoxifen and metabolites.

Tamoxifen and metabolite concentrations in serum and breast cancer tissue during three dose regimens in a randomized preoperative trial.

PURPOSE: Both therapeutic and adverse effects of tamoxifen may be related to its tissue concentrations. We investigated concentrations of tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen, and N-didesmethyltamoxifen in serum, normal breast, and breast cancer tissues during conventional dosage and two low-dose regimens. Furthermore we studied tamoxifen effects on the cancer proliferation marker Ki-67, and on sex hormone-binding globulin (SHBG). EXPERIMENTAL DESIGN: From September 1999 to August 2001, 120 breast cancer patients were randomized to 20-, 5-, or 1-mg tamoxifen daily. We measured serum and tissue concentrations of tamoxifen and three metabolites after 28 days of treatment, and the changes between baseline and post-treatment levels of SHBG and Ki-67. RESULTS: The median (range) tamoxifen concentrations (ng/ml) at doses of 1, 5, and 20 mg daily (n = 38, 37, and 36) were 7.5 (2.9-120.9), 25.2 (1.9-180.9), and 83.6 (8.7-134.4) in serum, and 78.2 (35.9-184), 272.3 (122-641), and 744.4 (208.6-2556) in breast cancer tissue, respectively. Tamoxifen levels followed a dose-concentration relationship. The concentrations of tamoxifen and metabolites were related to each other. Serum and tissue concentrations of tamoxifen were associated with corresponding changes of SHBG levels, whereas changes of Ki-67 levels were not related. CONCLUSIONS: Estrogen agonistic effects of tamoxifen on SHBG decreased with lower dosage, whereas tamoxifen effects on Ki-67 expression did not change. This together with a >10-fold variation in serum tamoxifen concentrations and a serum to tissue concentration relationship suggest that tamoxifen treatment may be improved by administration of lower doses and therapeutic drug monitoring.

Estrogens Correlate with PELP1 Expression in ER Positive Breast Cancer.

The Proline-, glutamic acid- and leucine-rich protein 1 (PELP1) is an estrogen receptor (ER) coactivator and a proto-oncogene known to be deregulated in endocrine cancers. In breast cancer, PELP1 overexpression has been associated with endocrine therapy resistance. Although PELP1 is known to be regulated by estrogens in vitro, its association with estrogen levels within the tissue of breast cancer patients has not previously been assessed. Here, we determined PELP1 mRNA expression levels in paired samples of normal and malignant breast tissue obtained from 32 postmenopausal and 11 premenopausal women. In the total sample set, PELP1 levels were higher in tumors compared to normal breast tissue (P = 0.041). Among postmenopausal women, PELP1 tumor levels correlated positively with estrone (E1) and estradiol (E2) levels in both normal tissue (r = 0.543, P = 0.003 and r = 0.601, P = 0.001, respectively) and plasma (r = 0.392, P = 0.053 and r = 0.403, P = 0.046, respectively). Analyzing all ER+ tumors (n = 26), PELP1 correlated positively with E1 and E2 in tumor tissue (r = 0.562, P = 0.003 and r = 0.411, P = 0.037, respectively) and normal tissue (r = 0.461, P = 0.018 and r = 0.427, P = 0.030, respectively) in addition to plasma E1, E2 and estrone sulphate (E1S) concentrations (r = 0.576, P = 0.003, r = 0.456, P = 0.025 and r = 0.406, P = 0.049, respectively). Finally, PELP1 correlated positively with ER mRNA (ESR1) (r = 0.553, P = 0.026) in ER+ tumors, whereas a negative association between PELP1 and ESR1 (r = -0.733, P = 0.010) was observed in ER- breast tumors. Taken together, tumor PELP1 mRNA expression is associated with estrogen levels in breast cancer, suggesting a potentially important role of PELP1 in ER+ breast cancer growth in vivo.

The active tamoxifen metabolite endoxifen (4OHNDtam) strongly down-regulates cytokeratin 6 (CK6) in MCF-7 breast cancer cells.

INTRODUCTION: Tamoxifen is an anti-estrogen drug used in treatment of Estrogen Receptor (ER) positive breast cancer. Effects and side effects of tamoxifen is the sum of tamoxifen and all its metabolites. 4-Hydroxytamoxifen (4OHtam) and 4-hydroxy-N-demethyltamoxifen (4OHNDtam, endoxifen) both have ER affinity exceeding that of the parent drug tamoxifen. 4OHNDtam is considered the main active metabolite of tamoxifen. Ndesmethyltamoxifen (NDtam) is the major tamoxifen metabolite. It has low affinity to the ER and is not believed to influence tumor growth. However, NDtam might mediate adverse effects of tamoxifen treatment. In this study we investigated the gene regulatory effects of the three metabolites of tamoxifen in MCF-7 breast cancer cells. MATERIAL AND METHODS: Using concentrations that mimic the clinical situation we examined effects of 4OHtam, 4OHNDtam and NDtam on global gene expression in 17ß-estradiol (E2) treated MCF-7 cells. Transcriptomic responses were assessed by correspondence analysis, differential expression, gene ontology analysis and quantitative real time PCR (Q-rt-PCR). E2 deprivation and knockdown of Steroid Receptor Coactivator-3 (SRC-3)/Amplified in Breast Cancer 1 (AIB1) mRNA in MCF-7 cells were performed to further characterize specific effects on gene expression. RESULTS: 4OHNDtam and 4OHtam caused major changes in gene expression compared to treatment with E2 alone, with a stronger effect of 4OHNDtam. NDtam had nearly no effect on the global gene expression profile. Treatment of MCF-7 cells with 4OHNDtam led to a strong down-regulation of the CytoKeratin 6 isoforms (KRT6A, KRT6B and KRT6C). The CytoKeratin 6 mRNAs were also down-regulated in MCF-7 cells after E2 deprivation and after SRC-3/AIB1 knockdown. CONCLUSION: Using concentrations that mimic the clinical situation we report global gene expression changes that were most pronounced with 4OHNDtam and minimal with NDtam. Genes encoding CytoKeratin 6, were highly down-regulated by 4OHNDtam, as well as after E2 deprivation and knockdown of SRC-3/AIB1, indicating an estrogen receptor-dependent regulation.

Illness uncertainty in breast cancer patients: validation of the 5-item short form of the Mishel Uncertainty in Illness Scale.

PURPOSE: Several studies have shown that uncertainty about disease and fear of disease progression affects psychosocial adjustment and quality of life. The purpose of this study was to validate a Norwegian short version of the "The Mishel Uncertainty in Illness Scale" (SF-MUIS) and to examine the impact of uncertainty in illness in breast cancer patients. METHOD AND SAMPLE: 209 patients in breast cancer treatment completed questionnaires for SF-MUIS, Hospital Anxiety and Depression Scale (HADS), the Functional Assessment of Cancer Therapy-Breast (FACT-ES), and eight questions concerning quality of the patient information provided (IQP). Relationship between scores on uncertainty in illness and anxiety, depression, social support, emotional well-being, the quality of patient information provided, and age were studied by multiple regression analyses. RESULTS: Ordinal coefficient alpha for the Norwegian version of SF-MUIS was 0.70. Scores on SF-MUIS correlated significantly with scores on HADS (P = 0.001), FACT-ES (P = 0.001), and IQP (P = 0.001) indicating good convergent validity. The patients reported a moderate degree of uncertainty in illness. However, those who had been diagnosed with breast cancer for a year, reported higher scores than those newly diagnosed (P=<0.0001). Information provided was the sole significant predictor of illness uncertainty (P=<0.0001). CONCLUSION: The results of the present study confirm that the Norwegian version of the SF-MUIS is a suitable tool for assessment of uncertainty in breast cancer patients, who reported a moderate degree of uncertainty in illness.

Changes in adipose glucocorticoid metabolism before and after bariatric surgery assessed by direct hormone measurements.

OBJECTIVES: Increased intra-adipose cortisol is thought to promote obesity, but few human studies have investigated intra-adipose glucocorticoid hormones and none have demonstrated prospective changes with fat loss. DESIGN AND METHODS: Subcutaneous adipose tissue (SAT) was obtained from obese subjects before and 1-year after surgery-induced fat loss, and from nonobese controls. In a second similar cohort of obese subjects, adipocytes and stromal-vascular fraction were isolated. Intra-adipose cortisol and cortisone levels were analyzed by liquid chromatography mass spectrometry and HSD11B1/HSD11B2 mRNA by qPCR. RESULTS: SAT cortisol/cortisone ratio before fat loss, median 4.8 (interquartile range, 4.1-5.7), was higher than after fat loss, 1.9 (1.0-2.7) (P = 0.001), and compared to nonobese controls, 3.2 (2.4-3.9) (P = 0.005). Cortisone before fat loss, 2.3 (1.2-2.9) nmol/kg, was lower than after fat loss, 5.8 (3.0-10.2) nmol/kg (P = 0.042), and compared to controls, 5.1 (3.8-6.7) nmol/kg (P = 0.013). HSD11B1 was predominantly expressed in mature adipocytes, whereas HSD11B2 was expressed at a higher level in stromal-vascular fraction. CONCLUSIONS: The intra-adipose glucocorticoid metabolism was markedly altered in the extremely obese state with increased cortisol levels relative to cortisone, whereas fat loss restored this balance approximating nonobese subjects. Changes were more pronounced for cortisone than cortisol, suggesting an adaptive response to insufficient intra-adipose cortisol levels in obesity.

Functional polymorphisms in UDP-glucuronosyl transferases and recurrence in tamoxifen-treated breast cancer survivors.

BACKGROUND: Tamoxifen is oxidized by cytochrome-P450 enzymes (e.g., CYP2D6) to two active metabolites, which are eliminated via glucuronidation by UDP-glucuronosyl transferases (UGT). We measured the association between functional polymorphisms in key UGTs (UGT2B15*2, UGT2B7*2, and UGT1A8*3) and the recurrence rate among breast cancer survivors. METHODS: We used the Danish Breast Cancer Cooperative Group registry to identify 541 cases of recurrent breast cancer among women with estrogen receptor-positive tumors treated with tamoxifen for at least 1 year (ER(+)/TAM(+)), and 300 cases of recurrent breast cancer among women with estrogen receptor-negative tumors who were not treated with tamoxifen (ER(-)/TAM(-)). We matched one control to each case on ER status, menopausal status, stage, calendar period, and county. UGT polymorphisms were genotyped from archived primary tumors. We estimated the recurrence OR for the UGT polymorphisms by using logistic regression models, with and without stratification on CYP2D6*4 genotype. RESULTS: No UGT polymorphism was associated with breast cancer recurrence in either the ER(+)/TAM(+) or ER(-)/TAM(-) groups [in the ER(+)/TAM(+) group, compared with two normal alleles: adjusted OR for two UGT2B15*2 variant alleles = 1.0 (95% CI, 0.70-1.5); adjusted OR for two UGT2B7*2 variant alleles = 0.96 (95% CI, 0.65-1.4); adjusted OR for one or two UGT1A8*3 variant alleles = 0.95 (0.49-1.9)]. Associations were similar within strata of CYP2D6*4 genotype. CONCLUSIONS: Functional polymorphisms in key tamoxifen-metabolizing enzymes were not associated with breast cancer recurrence risk. IMPACT: Our results do not support the genotyping of key metabolic enzyme polymorphisms to predict response to tamoxifen therapy.

Effect of low-dose tamoxifen on steroid receptor coactivator 3/amplified in breast cancer 1 in normal and malignant human breast tissue.

PURPOSE: Nuclear receptor coactivator expression and activity may partly explain the complex agonist/antagonist effects of tamoxifen at clinical level. In a preoperative trial, dose reduction from 20 to 1 mg tamoxifen was associated with retained antiproliferative effect on breast cancer. Here, we assessed the gene expression of the steroid receptor coactivators SRC-1, SRC-2/transcription intermediary factor 2, and SRC-3/amplified in breast cancer 1 (AIB1) and the growth factor receptor HER-2/neu under three tamoxifen dose regimens. EXPERIMENTAL DESIGN: Surgical specimens from estrogen receptor-positive breast cancer and adjacent normal breast tissue from 64 patients treated 4 weeks preoperatively with 20, 5, or 1 mg/d tamoxifen and 28 nontreated breast cancer controls were analyzed for coactivator and HER-2/neu mRNA expression using real-time reverse transcription-PCR. The gene expression levels were related to immunohistochemical expression of Ki67, serum levels of insulin-like growth factor I and sex hormone binding globulin, other prognostic factors, and clinical outcome. RESULTS: The coactivators and HER-2/neu mRNA levels were higher in malignant compared with normal tissue (P < 0.001). Tamoxifen significantly increased the expression of coactivators in normal and malignant tissue irrespective of dose, especially for SRC-3/AIB1 (P < 0.001 tamoxifen-treated versus nontreated subjects). SRC-3/AIB1 and HER-2/neu mRNA levels were positively correlated (P = 0.016), but the coactivators could not explain the variability of Ki67, insulin-like growth factor I, and sex hormone binding. Although not significant, SRC-3/AIB1 tended to be higher in subjects with poor clinical outcome and unfavorable prognostic factors. CONCLUSIONS: Increased coactivator mRNA levels seem to be an early response to tamoxifen without dose-response relationship in the 1- to 20-mg range. Clinical and molecular effects of low-dose tamoxifen should be further explored.