Breast cancer is a multidisciplinary disease.¹ Over 2.3 million incidences in both sexes combined and 685,000 deaths from breast cancer occurred across ethnicities in 2022.² It is a major public health concern in Algeria, with an incidence rate of about 11847 new cases in 2018. Approximately 75% of breast cancer patients are estrogen receptor-positive (ER+)³, making this hormone a target of endocrine therapies (Ets). Tamoxifen has been approved by the Food and Drug Administration (FDA) for the treatment of ER+ breast cancer, and several studies are currently investigating its therapeutic potential. Tamoxifen undergoes metabolism via cytochrome p450 enzymes in the liver, with the major metabolites formed being endoxifen and 4-hydroxytamoxifen, which are believed to be more potent anti-estrogens than tamoxifen itself.^4,5 The main pathway for tamoxifen biotransformation is via its hydroxylation to form 4-hydroxytamoxifen, and then to endoxifen, which is catalyzed primarily by Cytochrome P450 2D6 (CYP2D6). To standardize genotype-to-phenotype translations of the various CYP2D6 variant alleles, the mechanisms for assigning enzyme activity scores were established by Gaedigk and colleagues.^6,7. In this system, CYP2D6 variant alleles were assigned enzyme activity scores between 0 and 1, referring to no (‘null’) and fully functional enzyme activities, respectively. Based on the assigned activity score of the alleles, individual diplotypes’ scores are calculated, ranging from ‘0’ for null/null carriers to ‘≥3’ for carriers of multiplications of fully functional alleles.^8-11 The metabolism of tamoxifen is altered by CYP2D6 polymorphisms, which can change a drug’s side effects profile and treatment efficacy depending on the CYP2D6 phenotype. Poor Metabolizers (PMs) are at an elevated risk for failure to reach therapeutic levels.¹² Some of the negative side effects of tamoxifen can be attributed to its agonist or antagonist actions, depending on the target tissue and the presence or absence of co-activators or co-repressors.¹³ Therefore, they are often the reason for discontinuation of the treatment, which would otherwise be potentially lifesaving.¹⁴ Potentially through the agonist action on the uterus, tamoxifen is known to cause uterine fibroids, post-menopausal vaginal bleeding, and menstrual changes. Otherwise, fatty liver is a common side effect of this drug that affects about 43% of patients¹⁵ within the first two years of treatment.^16-17 CYP2D6 polymorphisms and the risk of side effects during tamoxifen treatment have been the subject of prior research.^18-20 In the current study, we assessed the association between CYP2D6 polymorphisms and tamoxifen efficacy in the Algerian population receiving tamoxifen as adjuvant therapy in ER+ breast cancer.

Table 3 shows the frequency distribution of CYP2D6 phenotype among breast cancer patients. In total, 9 different alleles within 25 genotypes were identified. A total of 29 patients (38.1%) had a multiplication of the CYP2D6 allele. The most frequent CYP2D6 gene polymorphism for ultra-rapid metabolizer (URM) was *2/*2XN, which occurred in 14 patients (18.4%). Also, 40 patients (52.6%) showed a normal metabolizer (NM) phenotype (CYP2D6*1, CYP2D6*2, CYP2D9*35, and CYP2D6*39), with the most common allele being *1/*2 with a frequency of 13 (17.1%). PM (CYP2D6*5) and intermediate metabolizer (IM) (CYP2D6*10, CYP2D6*17, and CYP2D6*41) were seen in 1 (1.3%) and 6 (7.9%) patients, respectively. Two-sided Fisher’s exact test was used to determine the association between the Clinical parameters and CYP2D6 diplotype. Significant differences between tamoxifen’s adverse effects and different groups of CYP2D6 diplotype were observed (P<0.001).

Tamoxifen’s adverse effects were evaluated in all patients. Table 4 indicates the clinical parameters in association with diplotype. Significant differences between tamoxifen adverse effects and different groups of CYP2D6 diplotype were observed (P<0.00). No adverse effect was found in 47 (61.8%) of the patients, and a positive adverse effect was found in 29 (38.1%) of the study group. Diabetes mellitus was detected in n=8 (10.5%) of patients, those in groups categorized as NM/IM n=6 (7.8%), URM/NM/ n=1 (1.3%), and NM/PM n=1(1.3%) (P<0.00). However, hyperlipidemia was found in n = 8 (10.5%) of patients, those in groups categorized as NM/IM n=3 (3.9%), URM/NM n=1 (1.3%), NM/NM n=1 (1.3%), NM/PM n=1 (1.3%), IM/IM n=1 (1.3%) and PM/PM n=1 (1.3%) (P<0.00). Regarding hypertension, 11 patients (14.8%) developed hypertension after the commencement of tamoxifen treatment. Specifically, 4 patients had NM/IM diplotype (5.2%), 2 patients showed NM/NM (2.6%), 2 patients had PM/IM (2.6%), 1 patient showed URM/URM (1.3%), 1 patient had URM/NM (1.3%), and 1 patient showed PM/PM (1.3%) (P<0.00). Abdominal ultrasound identified hepatosteatosis in 20 (26.3%) patients, while no hepatosteatosis was detected in 56 (73.6%) patients. Also, 13 patients (17.1%) had fatty liver following the commencement of tamoxifen therapy diagnosed as NM/IM, 2 (2.6%) were URM/NM, 2 (2.6%) were IM/IM, 2 (2.6%) were IM/PM and 1 (1.3%) was PM/PM. We observed a sequential association between the initiation of tamoxifen therapy and the occurrence of fatty liver in patients performing at least one copy of deficient enzyme activity (Figure 1). Of patients who developed fatty liver, 8 (40%) had Diabetes mellitus, 6 (30%) had hyperlipidemia, and 8 (40%) had hypertension.

Data on patients who experienced recurrences were obtained retrospectively from medical records. Table 5 illustrates the association between CYP2D6 diplotype and recurrence in combination with plasma levels of tamoxifen metabolites. In our cohort, 7 (9.2%) of the population had disease relapse, 1 patient was PM/PM (1.3%), 1 patient was NM/PM (1.3%), 2 patients (2.6%) were IM/IM, and3 (3.9%) patients were IM/PM. The functional allele CYP2D6*1 served as the reference group.

The relationship between plasma endoxifen concentration and experiencing recurrence is presented in Figure 2. It was found that the combination genotype NM/PM (P=0.03), IM/IM (P=0.02), IM/PM (P<0.001), and PM/PM (P<0.001) was more strongly related to disease recurrence than NM carriers of the function allele.

Tamoxifen is a prodrug indicated in the treatment of all stages of ER+ breast cancer. Pharmacogenetic differences are considered an important factor in oncology because of the potential consequences of toxicities due to the high frequency of genetic inter-variability. Numerous studies have been conducted to investigate the relationship between CYP2D6 polymorphisms and tamoxifen metabolism, and the role of tamoxifen in increasing the risk of breast cancer adverse events and recurrence. CYP2D6 mutations have a major impact on the appearance of side effects in patients treated with tamoxifen.^23-28 In our study, we investigated the impact of CYP2D6 polymorphisms on developing side effects and recurrences in pre-menopausal women with ER+ breast cancer treated with tamoxifen as the adjuvant therapy.

Our study showed a significant association between the presence of a defective copy of enzyme activity and the development of tamoxifen adverse effects after the commencement of tamoxifen therapy. During the examination of clinical records, the patients were found to have normal hepatic status before starting tamoxifen. Our study showed a sequential association between the initiation of tamoxifen therapy and the occurrence of fatty liver in patients with at least one copy of deficient enzyme activity. Additionally, our results revealed that a low plasma endoxifen was observed in patients categorized as NM/PM but also in patients with two reduced functional alleles (IM/ IM), patients with one reduced functional allele in combination with one null allele (IM/PM) or patients with two null alleles (PM/PM), implying that the absence of enzyme activity or a decrease in its level is closely related to potentially sub-therapeutic endoxifen levels as well as developing adverse effect of tamoxifen.

As is well-documented, CYP2D6 is involved in the conversion of tamoxifen to 4-hydroxytamoxifen, which is subsequently oxidized to endoxifen.^25,29 However, tamoxifen has a potential impact on mitochondria; it causes mitochondrial dysfunction 17,19-20 accordingly, the inhibition of mitochondrial β-oxidation, and the accumulation of fatty acids, which are converted to triglycerides, resulting in hepatic steatosis. ³⁰ Nevertheless, endoxifen has less impact on mitochondria when compared to tamoxifen. ³⁰ Consequently, patients with lower levels of endoxifen and those with decreased enzyme activity are more likely to experience mitochondrial dysfunction and develop fatty liver. These findings may elucidate the considerable frequency of higher levels of fatty liver detected in our population among patients categorized as CYP2D6 NM/PM, IM/IM, IM/PM, or PM/PM diplotypes.

Moreover, we also aimed to investigate the relationship between endoxifen plasma levels and the occurrence of recurrences, particularly among patients developing adverse effects. The results indicated that patients with increased plasma endoxifen concentrations were significantly less likely than patients with reduced or null activity to report recurrences (P < 0.05); such patients were categorized as PM/PM, IM/IM, IM/PM, and NM/PM. Thus, patients with at least one adverse effect of tamoxifen were positively associated with increased rates of recurrence than other genotypes during treatment. These findings revealed that tamoxifen efficacy and minor rates of adverse effects are correlated with a certain level of endoxifen concentration in plasma. The large-scale production of the potent metabolite endoxifen can explain this trend toward lower recurrence rates; this partly clarifies the main role of endoxifen in the repression of tumor cells. We could show in concordance with previous studies^31,32 that genotypes associated with normal or increased CYP2D6 activity lead to a favorable treatment outcome under tamoxifen. Our results are in agreement with a case-control study involving 46 women with breast cancer and 136 controls, where it was found that the frequency of CYP2D6*4 was higher in patients (9%) than in controls (1%) (P=0.01). However, contradictory results have been reported from two studies in the US and Sweden by Owen et al.³³, finding no association between CYP2D6*4 and the response to tamoxifen or recurrence of breast cancer.

Our results show that CYP2D6 polymorphism should be considered in predicting the occurrence of adverse effects of fatty liver in women treated with tamoxifen. Alternative treatment can be considered, and lifestyle modifications can be implemented. Although CYP2D6 has a moderate capacity for drug metabolism in the liver, it is highly polymorphic and, therefore, may alter the metabolism of tamoxifen toward the activation pathways. Breast cancer patients with CYP2D6 NM/PM, IM/IM, PM/PM, and IM/PM diplotypes may benefit less from tamoxifen treatment. Consequently, they are more likely to experience the adverse effects of tamoxifen and disease recurrence.

This study was approved by the ethics committee of Dr BENBADIS - Constantine University Hospital Centre and complied with the guidelines laid down in the Declaration of Helsinki (1964). All procedures involving human subjects were approved by the local ethical committee of Dr. BENBADIS – Constantine University Hospital Centre. Informed consent was obtained from individual study participants or their families before being recruited for the study.