Tamoxifen in the treatment of luminal breast cancer. Implications of active metabolites on gene expression, side effects and clinical outcome

Abstract

Estrogen is a driver for the development and progression of luminal breast cancer, and up to 80% of all breast cancers diagnosed belong to the luminal subtype. The estrogenic effect on breast tumors can be reduced by the adjuvant use of the antiestrogenic drug tamoxifen, which has significantly improved outcomes in luminal breast cancer patients over the last several decades. The 10-year recurrence rate in luminal breast cancer patients is approximately 25%. Thus, some patients do not achieve the desired curative effect of adjuvant tamoxifen. Tamoxifen is regarded as a weak anti-estrogen, and the anti-estrogenic effect is believed to be dependent on its active metabolites endoxifen and 4OHtam, which have up to 100-fold higher affinities to the estrogen receptor (ER) compared to tamoxifen itself. Worryingly, a significant subgroup of patients has a reduced ability to metabolize the drug, which translates into lower concentrations of the important active metabolites. Studies have indicated that these patients derive suboptimal therapeutic effects from tamoxifen. In addition, a large number of patients using tamoxifen discontinue treatment before the predetermined treatment time, which may leave these patients at higher risk of relapse. Therefore, increasing knowledge about the function of and determining possible therapeutic thresholds for the active metabolites represent promising potential progress towards individualized tamoxifen treatment and thereby improve patient outcomes. Identifying biomarkers that predict tamoxifen discontinuation may also be used to further tailor individual treatment. In paper I, we performed microarray global gene expression analyses on NDtam, 4OHtam and endoxifen-treated MCF-7 cells to elucidate the gene regulative roles of these metabolites. Global gene expression analyses revealed a step-wise regulation of genes in which endoxifen and 4OHtam resulted in the strongest and second strongest regulation of both up- and down-regulated genes, respectively. The change in global gene expression after treatment with NDtam was minimal. The two active metabolites regulated genes in similar gene ontology classes, implying a degree of similar function between the two metabolites. We also provided evidence for all three isoforms of CytoKeratin6 (CK6) being estrogen regulated, making CK6 a potential antiproliferative target of tamoxifen that should be researched further. In paper II, we retrospectively compared the predictive value of CYP2D6 phenotype groups and concentrations of active tamoxifen metabolite on long-term clinical outcome. Blood and serum from 99 operable breast cancer patients treated with 5-year adjuvant tamoxifen were analyzed for CYP2D6 genotypes and concentration levels of active tamoxifen metabolites, respectively. CYP2D6 phenotypes were found to be correlated to Z-endoxifen and Z-4OHtam concentrations. However, Kaplan-Meier analyses showed that CYP2D6 phenotypes were not associated with breast cancer specific-survival (BCSS). The same analyses were repeated using concentrations of tamoxifen metabolites; patients with concentrations below 9 nM and 3.26 nM for Zendoxifen and Z-4OHtam, respectively, had significantly worse BCSSs compared to patients with concentrations above these cut-off points. When we included a third cutoff point, representing patients with high concentration levels found in CYP2D6 ultrarapid metabolizers, we found that these patients had no BCSS endpoints up to 19 years after surgery. The BCSS results from these three groups also translated into overall survival. Our findings indicate that the concentrations of active tamoxifen metabolites may be used to predict the therapeutic effects of tamoxifen. In paper III, we investigated the association between tamoxifen metabolites and side effects in 220 patients that delivered blood samples and patient reported outcome measures yearly from 2011 to 2017. Analyses of side effects revealed hot flashes, vaginal atrophy symptoms, and joint pain to be most prevalent. Association analyses demonstrated that patients with high levels of tamoxifen, Z-4’OHtam and tamNoX were more likely to report vaginal dryness than patients with lower levels. Our secondary objective was to compare discontinuation rates obtained through the Norwegian prescription database to rates determined by longitudinal drug monitoring of systemic tamoxifen concentrations. Drug monitoring showed that only 6% of patients were not taking tamoxifen during a period that was covered by a tamoxifen prescription. Our results indicate that drug monitoring of tamoxifen metabolites may be used as biological predictor of vaginal dryness and that discontinuation rates obtained through pharmacy refill registries represent valid results.