Role of Cytochrome P450 Polymorphism in Activation and Metabolism of Tamoxifen in ER Positive Breast Cancer Patients

Abstract

Breast cancer remains the most prevalent cancer among women worldwide. Tamoxifen or aromatase inhibitors are recommended in endocrine therapy because most newly diagnosed breast cancer cases are estrogen-receptor positive. After two or three years of endocrine therapy, tamoxifen has been recommended as either a monotherapy or in combination with an aromatase inhibitor. When administered adjunctively, tamoxifen decreases breast cancer mortality and relapse rates; however, when combined with other metastatic breast cancer treatments, it has been shown to increase survival durations. It is unfortunate that tamoxifen's effectiveness varies significantly. The study objective was to investigate the CYP2C9(*2,*3), CYP2C19(*2,*3 &*17) CYP3A4(*22) and CYP3A5(*3,*6) frequency between Pakistani estrogen positive breast cancer patients and unrelated healthy controls, and also effect of gene variants on the pharmacogenetics and pharmacokinetics of tamoxifen in patients with estrogen positive breast cancer. ER-positive breast cancer patients receiving 20 mg/day of tamoxifen (n = 430) and healthy, unrelated individuals (n = 410; control group) comprised the study population. High-Performance Liquid Chromatography was utilized to determine the steady-state plasma concentrations of tamoxifen and its three metabolites in the patients. DNA was extracted and analyzed by ARMS PCR and AS-PCR followed by RFLP. Five phenotypes observed are as follows, extensive metabolizer (EM), poor metabolizer (PM), intermediate metabolizer (IM), Rapid Meabolizer (RM) and ultra-rapid metabolizer (UM). Further statistical analysis were performed on median plasma concentration of tamoxifen and derivatives. Plasma metabolic ratio and total metabolic ratio were determined and correlated with each genotype. In our population, CYP2C9*2 heterozygous (OR: 0.4; 95% CI: 0.53–0.56; p = 0.0001) and homozygous mutant (OR: 3,12; 95% CI: 1.80–5.43; P = 0.0001) condition were identified as breast cancer risk factors. The CYP2C9*2 gene variant had no significant effect on the metabolic ratio between tamoxifen and its three metabolites. However, an insignificant decrease was recorded in the median plasma concentration of 4-OH-Tam in the subjects having heterozygous (*1/*2) (P = 0.747) and mutant (*2/*2) (P = 0.223) genotypes. More than 65% of healthy individuals were extensive metabolizers (*1/*1) for CYP2C19, whereas more than 70% of ER-positive BC patients were rapid and ultra rapid metabolizers (*1/17*, *17/17*). The polymorphism CYP2C19*17 is significantly x Abstract associated with higher 4-OH-Tamoxifen. Patients with the *17/*17 genotype exhibited 1 to 1.5-fold higher 4-OH-Tamoxifen, which was also high in patients with the *1/*2 and *2/*2 genotypes. Which suggests that CYP219*17 has a significant effect on the higher production of 4-OH-Tamoxifen. The allele9frequencies of the CYP3A4*22 variant in ER positive breast9cancer patients and healthy controls, demonstrated no noteworthy difference between the allele frequencies of CYP3A4*22. Plasma metabolic ratios of tamoxifen and its metabolites indicates that CYP3A4*22 does not have any significant effect on the metabolism of tamoxifen and metbolities. CYP3A5*3 and *6 genotyping results for unrelated healthy individuals and ER positive breast9cancer patients in Pakistani population indicates that most of the individuals in both the groups belongs to extensive metabolizer. The frequencies of the CYP3A5 alleles did not differ noticeably from one another. Our study demosttrates that CYP3A5 do not have any significant impact on the metabolism of tamoxifen and its metabolites. Tamoxifen and its metabolites were tested for their biological effects with different CYP2C19*17 genotypes (EM, RM and UM) on cell proliferation and estrogen responsive gene regulation in the MCF-7 breast cancer cell line. Real clinical levels of tamoxifen metabolites in breast cancer patients, as well as actual amounts of estrogens reported in premenopausal individuals receiving tamoxifen, were employed in vitro. Interestingly, tamoxifen anaits primary metabolites were not able to fully inhibit the estrogen-stimulated expression of estrogen-responsive genes in MCF-7 cells (P < .05 for all genes), but the addition of endoxifen was able to produce additional antiestrogenic effect on these genes. Our results suggest that the circulating levels of tamoxifen and its metabolities are not sufficient enough to completely block the estrogen stimulated growth in cell line.