Abstract Background and rationale: Side effects of adjuvant treatment with tamoxifen (tam) may impair Quality of Life (QoL) and have been suggested as an independent variable for discontinuation of tam1. There are large inter-patient variabilities in prevalence and severance of side effects among tam users. Therefore, there is a need for biological markers that can predict side effects. A potential biological predictor is the serum concentrations of tam and/or its metabolites. In this prospective observational study we have analyzed serum concentrations of tam and 9 metabolites over 3 years. Patients Reported Outcome Measures (PROM) were obtained to elucidate possible associations between side effects, adherence and tam metabolism. Methods: Breast cancer patients using adjuvant tam (20mg/d) were recruited through the Prospective Breast Cancer Biobank project between 2011 and 2016. Inclusion criteria were ER positive status, ≥ 6 months tam use, tumor size of ≥0.1 cm and being able to read and write Norwegian. Concentration levels of tam and metabolites in serum were analyzed by LC-MS/MS2 and adherence data were collected through the Norwegian prescription database. PROM-data comprised of validated questioners reporting side effects of endocrine treatment and QoL. Statistical analyses comprised non-parametric tests, logistic regression, chi square tests and the Benjamin-Hochberg procedure to correct for multiple testing. Results: Associations between metabolite concentrations and side effects were run as a cross sectional analysis (N=149) and separate analysis of each year of follow-up with 85, 77 and 65 patients at the 1st, 2nd and 3rd year respectively. We found that 78 % of patients reported side effects, 66 % reported mood swings, 21 % reported severe hot flushes and 71 % reported decreased libido. When analyzing years separately, we found that on the 2nd year patients experiencing vaginal dryness had significantly higher levels of tamoxifen (P=0.032, after correction for multiple testing and adjustment for clinical relevant variables) compared to patients not experiencing vaginal dryness. Also, on year 2 the patients in the lower quartile of Z-endoxifen (≤17.9 nM) had significantly lower libido (p=0.015) compared to patients with Z-endoxifen levels >17.9 nM after adjustment for clinical relevant variables and correction for multiple testing. Analyses regarding adherence are not complete and more results will be presented in the poster. Discussion: Our data indicates that high serum concentrations of tam and low concentrations of Z-endoxifen are associated with vaginal dryness and sexual dysfunction. Patients reporting “very low libido” had the highest levels of tam, suggesting that slow metabolic conversion and accumulation of tam may contribute to sexual dysfunction. Our results were only significant in the second year of follow-up, possibly because patients wait to resume sexual activity after diagnosis, chemo and surgery. After receiving advice (i.e. lubricants), the symptoms are often reduced in the subsequent follow-up (3rd year). In conclusion, our results indicate that monitoring tam serum concentrations may be used to predict side effects. 1 Owusu C. et al. JCO. 2008 2 Helland T. et al. BCR. 2017 Citation Format: Helland T, Haugstøyl ME, Hagen KB, Kvaløy JT, Lunde S, Lode K, Lind RA, Gripsrud BH, Bifulco E, Gebreslase NS, Jonassen J, Hustad SS, Aas T, Lende TH, Lien EA, Janssen EA, Mellgren G, Søiland H. Serum concentrations of tamoxifen and Z-endoxifen may predict sexual dysfunction in the 2nd year of adjuvant endocrine treatment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-14-08.

Abstract Background Tamoxifen (tam) is the main adjuvant endocrine treatment option in premenopausal breast cancer (BC) patients comprising luminal-like tumors. However, a significant proportion of tam-users will experience a relapse within 15 years of primary surgery. We postulate that some patients do not achieve the full clinical benefit of tam due to inter-individual differences in the metabolism of the drug and that the clinical relevance of this may be different between molecular subtypes of BC. Here, we have compared the prognostic value of threshold levels of active tam metabolites in PAM50 luminal (lum) A and B molecular subtypes. Material and Methods A number of 64 lum-like BC patients who were relapse-free 3 years after surgery, were retrospectively analyzed in the observational Oslo1 study. All patients received 20 mg tamoxifen daily for 5 years. Serum was obtained at the time of the 3 years follow-up. A sensitive and accurate LC-MS/MS method was developed and validated for the detection and quantification of tam and 9 metabolites in human serum. The median follow-up time from serum sampling to BC death or last follow-up was 13.9 years (0.6-16.5). Recurrence score and molecular subtype of the patients were determined on FFPE-tumor samples using the PAM50 classification algorithm. Results A linear trend was identified for the correlation between active metabolite Z-4OHtam and BCSS (p=0.021, HR=0.64, CI95=0.43–0.93). There was no linear association between the remaining metabolites and BC outcome. We further explored the possible association between survival and concentration thresholds for the active metabolites Z-4OHtam and Z-endoxifen and identified supervised cut off values representing low concentrations for Z-4OHtam (≤3.26 nM) and Z-endoxifen (≤9.00 nM). BC patients with low Z-4OHtam had a BCSS of 33.3% compared to 82.8% in patients with Z-4OHtam >3.26 nM (p<0.001, logrank; HR=6.83, CI 95=2.09-22.36). Lum status (A vs B; HR=5.50, CI95= 1.66-18.25) and Z-4OHtam concentration status (high vs low; HR=6.05, CI95=1.74-21.06) were the only factors left in the final multivariable model. A log-linear relationship between the ROR score and BCSS (p=0.002, HR=1.09, CI95=1.03–1.15) was identified after adjustment of clinically relevant variables and lum status was highly prognostic, (Lum A vs B; p=0.001, HR=5.2, CI=1.72-15.46). Therefore, we wanted to compare the prognostic value of the Z-4OHtam threshold in patients subgroups stratified by lum status. Low concentrations of Z-4OHtam were associated with poorer survival for patients in the lum B group only (HR=4.94, CI 95=1.16-21-02). For the lum A patients no significant association was found. Discussion Low levels (≤ 3.26 nM) of the active tam metabolite Z-4OHtam was associated with a poorer long-term outcome in tam-treated BC patients. However, when grouping patients according to the PAM50-based molecular subtype, this was only significant in patients belonging to the lum B subtype. Our results suggest that higher levels of active tam metabolites and thus better ER blockage are more important in the more aggressive lum B subtype. Citation Format: Helland T, Søiland H, Hustad S, Lash TL, Kvaløy JT, Renolen A, Borgen E, Bifulco E, Henne N, Lien EA, Mellgren G, Naume B, Janssen EA. Serum levels of the active tamoxifen metabolite Z-4OHtam is predictive of long-term survival in luminal B subtype of breast cancer patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-12-05.

Abstract Background The genetic polymorphisms of CYP2D6 determine its enzymatic activity thereby the pharmacokinetic velocity by which tamoxifen (tam) is converted into active metabolites. Combined analyses of tam metabolites compared to CYP2D6 type and long-term survival could allow prediction of tam response and personalization of therapies. The clinical importance of defining such subgroups in the era of the new long-term (10-year) tam treatment paradigm is actualized. Material and Methods From May 1995 to December 1998, patients were included in an observational micro-metastasis study in the Oslo region and treated according to the national guidelines at the time (1). Serum samples were drawn at 3-year follow-up from 356 relapse-free patients, 106 of these were treated with tamoxifen. The median follow up time for breast cancer death was 16.8 years (3.5-19.4). Serum samples were processed using protein precipitation with acetonitrile. An Aquity UPLC system was used to chromatographically separate 10 tam metabolites using a BEH C18 Phenyl column (100 x 2.1 mm, 1.7 μm particle size) that was developed with a water-methanol gradient containing 0.1% formic acid. The LC system was coupled to a Xevo TQ-S tandem mass spectrometer equipped with an atmospheric pressure photoionization source. The method was validated with respect to linearity, imprecision, accuracy, and functional sensitivity according to FDA guidelines. Results The new LC-MS/MS method separated the active Z-isomers of 4OHtam and 4OHNDtam (endoxifen) from its inactive Z'-isomers and E-isomers. Imprecision (intra and inter-day CV %) was within 10 % for target concentrations for all metabolites and accuracies were in the range 95-106%. The method was validated with serum samples from 42 breast cancer patients using 20 mg of tamoxifen. The endoxifen concentrations ranged from 0 to 90 nM, with a median value of 25 nM. The previous observed endoxifen level of 10 nM in poor metabolizers (2) was used as cut-off for the grouping of patients. The nil endoxifen (NE) group (< 0.1nM, n=14) or low-endoxifene (LE) group (0.1-10 nM, n=8) were grouped together. Univariate survival analysis did not show a significant association between breast cancer specific survival and endoxifen levels. (p=0.15; logrank and p=0.18;Breslow). However, for the period beyond 10-years of follow-up the breast cancer survival differed between the high endoxifene (HE) group and the NE+LE groups. For patients surviving the first 10 years the breast cancer specific survival was 94.2% vs. 77.8% for the HE and NE+LE groups respectively (p=0.020, logrank and p=0.017, Breslow, HR=4.5, CI 95=1.1-17.9). In the multivariate analysis endoxifen ≤/> 10 nM remained the only factor in the final model. Discussion We developed a new accurate and precise LC-MS/MS method for the measurement of 10 tamoxifen metabolites. Importantly, the method separates active and inactive isomers of 4OHtam and 4OHNDtam/endoxifen. Despite the low number of patients, we observed a poorer long-term survival beyond 10 years in patients with nil or low serum concentration of endoxifen. A comprehensive analysis is presented addressing the relationship between genotyped based and metabolite based prediction of long-term outcome in tamoxifen treated breast cancer patients. Ref: 1. Wiedswang G, et al. Clin Ca Res; 2004 2. Mürdter TE, et al. Clin Pharmacol Ther; 2011. Citation Format: Helland T, Henne N, Bifulco E, Hustad SS, Kristensen VN, Lash T, Borgen E, Janssen EAM, Lien EA, Naume B, Mellgren G, Søiland H. Metabolite-guided long-term prediction of outcome in tamoxifen treated breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-09-11.

Tamoxifen is an anti-estrogen drug used in the treatment of Estrogen Receptor (ER) positive breast cancer. 4-Hydroxy-N-demethyltamoxifen (4OHNDtam, endoxifen) is considered the main active metabolite of tamoxifen. In this study we investigated the gene regulatory effects of the three metabolites of tamoxifen in MCF-7 breast cancer cells.

Objective: Primary hyperparathyroidism (PHPT) has been associated with low-grade inflammation and increased risk of cardiovascular disease (CVD). The aim of the study was to investigate systemic levels of pro-inflammatory proteins that previously have not been examined in patients with PHPT. The selection of the pro-inflammatory biomarkers included in this study, MMP9, S100A4, S100A8/A9 and the receptors sCD14 and RAGE, was based on a previous microarray screen of mRNAs in adipose tissue from PHPT patients.Design: A prospective study was conducted on a total of 57 patients with PHPT and a control group of 20 healthy blood donors.Methods: PHPT patients with normalisation of serum calcium levels after parathyroidectomy were followed for 6 months. Forty-two patients participated in the longitudinal study, in which blood samples were taken at inclusion, and 1, 3 and 6 months after surgery.Results: We observed increased serum levels of MMP9 (P=0.029), S100A4 (P<0.001) and sCD14 (P=0.002) in the 57 patients with PHPT compared to the control-group. During 6 months of follow up, S100A4 (P=0.022) and sCD14 (0.002) decreased significantly, while serum levels of MMP9 increased (P=0.025).Conclusions: The results demonstrate an increased inflammatory response in PHPT patients shown by elevated MMP9, S100A4 and sCD14 at inclusion. During the 6 months of follow-up, MMP9 increased further, possibly due to the tissue repair process after parathyroidectomy. S100A4 and sCD14 decreased after surgery demonstrating a partial reversal of the systemic inflammation.

Introduction: Parathyroid hormone (PTH) and vitamin D are important hormones regulating calcium metabolism. In patients with primary hyperparathyroidism (PHPT) excessive amounts of PTH are produced. PTH influences the metabolism of vitamin D by increasing the activity of the 1α-hydroxylase as well as the 24-hydroxylase. Bone mineral density (BMD) is decreased and serum calcium levels elevated in PHPT patients.

Introduction: Tamoxifen dosage is based on the one-dose-fits-all approach. The anticancer effect of tamoxifen is believed to be due to the potent metabolite 4-hydroxy-N-desmethyltamoxifen (4OHNDtam, endoxifen). whereas the demethylated metabolites of tamoxifen have been associated with its side effects. The hydroxylated metabolites are converted from tamoxifen to a major part through the cytochrome P450 enzyme (CYP) 2D6. The inter-individual variation of the activity of this enzyme due to genetic polymorphisms may be a predictor of outcome during tamoxifen treatment due to influence on the concentration of the active metabolite 4OHNDtam (endoxifen). However, the results from clinical studies are partly contradictory. The conflicting results have been explained by differences in study designs, including size or different genetic models for the assessment of phenotypes.

Objectives Primary hyperparathyroidism (PHPT) has been associated with low-grade inflammation and elevated risk of cardiovascular disease (CVD). In inflammatory conditions, interferon-gamma (IFN-gamma) activity is enhanced and a decreased circulating concentration of vitamin B6 is often observed. Such changes in IFN-gamma activity or vitamin B6 levels have been associated with increased incidence of CVD. The aim of the study was to investigate systemic markers of IFN-gamma-mediated immune activation, such as neopterin, the kynurenine-to-tryptophan ratio (KTR) and kynurenine pathway metabolites, as well as B6 vitamers in patients with PHPT. Design/subjects A total of 57 patients with PHPT and a control group of 20 healthy blood donors were included in this study. PHPT patients who responded positively to parathyroidectomy were followed for 6 months. Forty-three patients participated in the longitudinal study in which blood samples were taken at inclusion and 1, 3 and 6 months after surgery. Results Plasma concentrations of the B6 vitamers pyridoxal 5'-phosphate (PLP) (P = 0.007) and pyridoxal (P = 0.013) were significantly lower in the patient group compared to healthy control subjects. An increase in the KTR indicated that the kynurenine pathway of tryptophan metabolism was altered in PHPT patients (P = 0.015). During the initial 6 months after surgery, levels of PLP (P < 0.001) and anthranilic acid (P < 0.001) increased significantly, whereas neopterin decreased (P = 0.018). Conclusions The results of this study demonstrate altered levels of vitamin B6 and the KTR in PHPT patients, both of which may reflect cellular immune activation. These abnormalities should be considered in relation to the increased risk of CVD previously observed in patients with PHPT.

Primary hyperparathyroidism (PHPT) has been associated with low-grade inflammation and elevated risk of cardiovascular disease (CVD). In inflammatory conditions, interferon-γ (IFN-γ) activity is enhanced and a decreased circulating concentration of vitamin B6 is often observed. Such changes in IFN-γ activity or vitamin B6 levels have been associated with increased incidence of CVD. The aim of the study was to investigate systemic markers of IFN-γ-mediated immune activation, such as neopterin, the kynurenine-to-tryptophan ratio (KTR) and kynurenine pathway metabolites, as well as B6 vitamers in patients with PHPT. A total of 57 patients with PHPT and a control group of 20 healthy blood donors were included in this study. PHPT patients who responded positively to parathyroidectomy were followed for 6 months. Forty-three patients participated in the longitudinal study in which blood samples were taken at inclusion and 1, 3 and 6 months after surgery. Plasma concentrations of the B6 vitamers pyridoxal 5′-phosphate (PLP) ( P  =   0.007) and pyridoxal ( P  =   0.013) were significantly lower in the patient group compared to healthy control subjects. An increase in the KTR indicated that the kynurenine pathway of tryptophan metabolism was altered in PHPT patients ( P  =   0.015). During the initial 6 months after surgery, levels of PLP ( P  <   0.001) and anthranilic acid ( P  <   0.001) increased significantly, whereas neopterin decreased ( P  =   0.018). The results of this study demonstrate altered levels of vitamin B6 and the KTR in PHPT patients, both of which may reflect cellular immune activation. These abnormalities should be considered in relation to the increased risk of CVD previously observed in patients with PHPT.

BACKGROUNDStudies have shown that Ki-67 response after short-term neoadjuvant aromatase inhibitors may predict recurrence in postmenopausal breast cancer, whereas its prognostic effect in premenopausal women is unknown.PATIENTS AND METHODSWe compared the prognostic and predictive value of baseline and post-treatment Ki-67 in 120 pre- and postmenopausal women with early-stage estrogen receptor-positive breast cancer who participated in a 4-week presurgical trial of tamoxifen.RESULTSAfter 7.2 years of follow-up, women with post-treatment Ki-67 in the second (14%-19%), third (20%-29%) and top (≥30%) quartiles had a recurrence hazard ratio of 2.92 [95% confidence interval (CI) 0.95-8.96], 4.37 (1.56-12.25) and 6.05 (2.07-17.65), respectively, as compared with those in the bottom quartile (<14%) (P-trend = 0.001). The risk of invasive disease recurrence was 2.2% (95% CI 0.9-5.0) per point increase in baseline Ki-67 (P-trend = 0.076) and 5.0% (95% CI 2.3-7.7) per point increase in post-tamoxifen Ki-67 (P-trend < 0.001). The risk of death was 5.5 (95% CI 1.26-23.16) times higher in patients with post-drug Ki-67 ≥20% than in those with Ki-67 <20% (P-trend = 0.006).CONCLUSIONSKi-67 response after short-term neoadjuvant tamoxifen is a good predictor of recurrence-free survival and overall survival, further supporting its use as surrogate biomarker to personalize adjuvant treatment and to screen novel drugs cost-effectively.

Pharmacogenetics has arrived in clinical psychiatric practice with the FDA approval of the AmpliChip CYP450 Test that genotypes for two cytochrome P450 2D6 (CYP2D6) and 2C19 (CYP2C19) genes. Other pharmacogenetic tests, including those focused on pharmacodynamic genes, are far from ready for clinical application. CYP2D6 is important for the metabolism of many antidepressants and antipsychotics, and CY2C19 is important for some antidepressant metabolism. Poor metabolizers (PMs), lacking the enzyme, account for up to 7% of Caucasians for CYP2D6 and up to 25% of East Asians for CYP2C19. Patients having three or more active CYP2D6 alleles (up to 29% in North Africa and the Middle East), are called CYP2D6 ultrarapid metabolizers (UMs). CYP2D6 phenotypes (particularly PMs) are probably important in patients taking tricyclic antidepressants (TCAs), venlafaxine, typical antipsychotics, and risperidone. The CYP2C19 PM phenotype is probably important in patients taking TCAs and perhaps citalopram, escitalopram, and sertraline. On the basis of the literature and the authors’ clinical experience, the authors provide provisional recommendations for identifying and treating CYP2D6 PMs, CYP2C19 PMs, and CYP2D6 UMs. The next few years will determine whether CYP2D6 genotyping is beneficial for patients taking the new drugs aripiprazole, duloxetine, and atomoxetine. Practical recommendations for dealing with laboratories offering CYP2D6 and CYP2C29 genotyping are provided.

Abstract Background: Biotransformation of tamoxifen leads to metabolites with different pharmacological and toxicological effects. Profiling and screening for active metabolites are important to assess their contribution to the overall therapeutic and adverse effects. The anticancer effect of tamoxifen is believed to be due to the hydroxylated metabolites 4hydroxytamoxifen (4OHtam) and 4hydroxyNdesmethyltamoxifen (4OHNDtam, endoxifen). Its demethylated metabolites have been related to side effects of the drug, whereas effects of tamoxifenNoxide (tamNoxide) have not been evaluated. In the present study we wanted to examine the distribution of tam and five of its metabolites in serum and normal and malignant breast tissues during normal dose (20 mg/day) and low dose (5 or 1 mg/day) regimens after 28 days of continuous dosing. Methods: Serum and biopsies from 56 patients were available for analysis. The concentrations of tamoxifen and the metabolites 4OHtam, 4OHNDtam, Ndesmethyltamoxifen (NDtam), Ndesdimethyltamoxifen (NDDtam), and tamNoxide in serum and tissues were measured by means of a recently developed highly sensitive liquid chromatography tandem mass spectrometry (LCMSMS) method. Results: Serum and biopsies from 56 patients were available for analysis. All compounds including the potent metabolites 4OHtam and 4OHNDtam were detected and measured in all samples. The tissue levels of tam and its hydroxylated and demethylated metabolites were highly related to the serum levels (p < 0.000 — 0.0069). The tumor tissue to serum concentration ratios of tamNoxide decreased by increasing doses (p < 0.0001) whereas this ratio was unchanged over the dose range 1-20 mg/day for the other compounds measured. Conclusion: Tamoxifen and its metabolites were detected in all samples examined including those sampled during the low dose regimens. The tissue levels of tamoxifen and its hydroxylated and demethylated metabolites were strongly corresponding to the serum concentrations, in contrast to those of tamNoxide. The strong relations between the concentrations of the potent active 4OHtam and 4OHNDtam (endoxifen) in serum and target tissues suggest that implementation of therapeutic drug monitoring may represent a tool to improve tamoxifen therapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-11-14.

Abstract We have previously shown in a window of opportunity, 3-arm, double-blind phase II randomized trial conducted in a single Institution that tamoxifen given for 4 weeks between tru-cut biopsy and surgery at a daily dose of 5 or 1 mg day was equivalent to 20 mg in decreasing ki-67 expression. A total of 31 premenopausal and 89 postmenopausal women with operable stage I-II ER-positive breast cancer were recruited (Decensi A et al, JNCI 2003;95:779-90). Here we analyzed the prognostic and predictive significance of ki-67% after a median follow-up of 86 months (11-111) in the same cohort of 120 women who underwent adjuvant treatment according to St. Gallen guidelines. At the current follow-up time DFS was 76% and OS was 92%. The median (IQ range) post-treatment ki-67% value was 12% (7-17) in women free of recurrence versus 20% (11-29) in women who relapsed (p=.0014). Post-treatment (surgery) ki-67% was positively associated with worse prognosis in univariate analysis (log-rank test =.0009, with ki-67% categorized in three groups: <7 (n=24), 7-19 (n=63), ≥20 (n=29); Figure).Post-treatment ki-67% was significantly associated with breast cancer death as well (p=.005, ki-67% <20 versus ≥20). In multivariate Cox analysis of DFS, both pre- and post-treatment ki-67 used as continuous variables were significantly associated with a higher risk of recurrence independent of adjuvant treatment, estrogen receptor level and pre-surgical tamoxifen dose (p=.002 and .04, respectively). Women with post-treatment ki-67% in the middle tertile (7-19%) and top tertile (≥20%) had a HR=2.1 (0.5-9.7) and HR=6.7 (1.5-30.2) of recurrence compared with women in the bottom tertile, respectively. There was no significant difference among tamoxifen dose groups on recurrence or death. In multivariate Cox analysis of OS, both pre- and post-treatment ki-67 used as continuous variables were significantly associated with a higher risk of breast cancer death (p=.0453 and .003, respectively). The risk of breast cancer death was 5 times higher in women with post-treatment ki-67 >20% than in women with ki-67<20% (HR=5.26, 1.22-23, p=.03). Our findings indicate that ki-67 is a prognostic and predictive marker in women with operable ER-positive breast cancer. The modulation of Ki-67% after a few weeks of hormonal intervention before surgery is a promising surrogate biomarker for outcome prediction and provides the opportunity to screen active agents in a cost-effective manner. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 406.

BACKGROUND:Acquired resistance to endocrine therapy in breast cancer is poorly understood. Characterisation of the molecular response to aromatase inhibitors in breast cancer tissue may provide important information regarding development of oestrogen hypersensitivity. METHODS:We examined the expression levels of nuclear receptor co-regulators, the orphan nuclear receptor liver receptor homologue-1 and HER-2/neu growth factor receptor using real-time RT-PCR before and after 13-16 weeks of primary medical treatment with the aromatase inhibitors anastrozole or letrozole. RESULTS:mRNA expression of the steroid receptor co-activator 1 (SRC-1) and peroxisome-proliferator-activated receptor gamma co-activator-1alpha (PGC-1alpha) was correlated (P=0.002), and both co-activators increased during treatment in the patient group as a whole (P=0.008 and P=0.032, respectively), as well as in the subgroup of patients achieving an objective treatment response (P=0.002 and P=0.006). Although we recorded no significant change in SRC-3/amplified in breast cancer 1 level, the expression correlated positively to the change of SRC-1 (P=0.002). Notably, we recorded an increase in HER-2/neu levels during therapy in the total patient group (18 out of 26; P=0.016), but in particular among responders (15 out of 21; P=0.008). CONCLUSION:Our results show an upregulation of co-activator mRNA and HER-2/neu during treatment with aromatase inhibitors. These mechanisms may represent an early adaption of the breast cancer cells to oestrogen deprivation in vivo.

CYP2D6 and SULT1A1 genotypes may partly explain the wide inter-individual variations in the serum levels of tamoxifen and its metabolites. We propose that therapeutic drug monitoring should be included in studies linking CYP2D6 and SULT1A1 genotypes to clinical outcome.

503 Background: Tamoxifen (T) is one of the most widely used drug for the treatment and prevention of estrogen receptor (ER) positive breast cancer. Individual genotype can modulate treatment efficacy. T metabolism is catalyzed by multiple enzymes: CYP2D6 is the main mediator for endoxifen, one of the most potent T metabolites. SULT may also play a role in T activation and can modulate hormone levels involved in breast cancer risk. Within a chemoprevention trial, we correlated T and its main metabolite concentration with polymorphisms (SNPs) of CYP2D6 and SULT genes. Methods: Premenopausal women (n 235) were randomly assigned in a double-blind four-arm trial to receive T 5 mg/d, fenretinide 200 mg/d, both agents, or placebo for 2 years CYP2D6 (*4–6) and SULT1A1*3 frequencies were determined using an Allelic Discrimination Assay, following a protocol for real time polymerase chain reaction. In the subjects receiving T, we measured plasma level of the drug, and its metabolites (endoxifen, 4-hydroxy-T, N-desmethyl-T, N-desdimethyl-T and Tnoxide) using a fluorescence detector at 12 and 24 months. Results: SNPs analysis was performed in 118 women; we found 65% wild type (WT), 31% heterozygous (H) and 5% mutated (M) for CYP2D6. The SNP frequency of SULT1A was A1, 61%; SULT1A2 36% and SULT1A3 3%. At one year a positive correlation was observed between CYP2D6 M and plasma level of N- desmethyl-T, the mean plasma level being 54.6, 62.4 and 108.5 ng/ml in WT, H and M, respectively, p=.001 (p=.01 at 24 months). Endoxifen was slightly increased in the WT compared to M (15.4 vs 12.9 ng/ml). SULT polymorphisms did not show any correlation with the drug and metabolite levels. Conclusions: CYP2D6 M mediates an increased level of N- desmethylT. This observation supports the role of CYP2D6 as an important enzyme in T metabolism, but more than one cytochrome P450 isoenzymes is required. to identify good or poor T metabolizers. No significant financial relationships to disclose.