No association found between CYP2D6 genotype and early breast cancer events in tamoxifen-treated patients

Abstract

Background. CYP2D6 is considered the key enzyme in tamoxifen metabolism. Several studies have investigated the relationship between the CYP2D6 genotype and tamoxifen treatment outcome, with discrepant results. CYP2D6 inhibitor use, aromatase inhibitor use, and chemotherapy may account for some of the discrepancies. We examined the association between CYP2D6 genotype and early breast cancer events in tamoxifen-treated breast cancer patients, in relation to CYP2D6 inhibitor use, aromatase inhibitor use, and chemotherapy. Material and methods. Pre- and postoperative questionnaires on lifestyle and concomitant medications were completed by 634 primary breast cancer patients between 2002 and 2008, among whom 333 patients had ER-positive tumors and received tamoxifen. CYP2D6*3, *4, *6, *10 and *41 were genotyped. Information on clinical data, breast cancer events, and tumor characteristics was obtained from patients’ charts, population registries, the Regional Tumor Registry, and pathology reports. Results. Median follow-up was 4.9 years. Neither poor metabolizers (adjusted HR 0.50; 95% CI 0.07–3.82) nor intermediate metabolizers (adjusted HR 1.00; 95% CI 0.47–2.11) had an increased risk of early breast cancer events when compared with extensive metabolizers. CYP2D6 activity score (taking into account genotype and CYP2D6 inhibitor use) was not associated with early breast cancer events (LogRank, P_trend = 0.44). Conclusions. CYP2D6 genotype was not associated with tamoxifen treatment outcome, even when CYP2D6 inhibitor use, aromatase inhibitor use, or chemotherapy was taken into account. CYP2D6 genotype may be of minor importance for tamoxifen-treated patients in Scandinavia.

Breast cancer is the most common cancer among women [1]. Despite an overall five-year relative survival rate of almost 90% for breast cancer patients in Sweden [2], it remains the primary cause of cancer death among women worldwide [1]. Though tamoxifen is an effective anti-estrogen treatment for breast cancer patients with estrogen receptor (ER) positive tumors, around 30% of tamoxifen-treated women relapse despite five years of tamoxifen treatment [3]. Markers of tamoxifen treatment-resistance could allow for more personalized treatment, improving the prognosis for many women [4].

The cytochrome P2D6 (CYP2D6) enzyme is considered the key enzyme in transforming tamoxifen into one of its most abundant active metabolites, endoxifen [5]. Patients with two non-functional CYP2D6 alleles have lower plasma concentrations of endoxifen than patients with functioning alleles [6,7]. Hence, it has been proposed that CYP2D6 genotype may be associated with recurrence among tamoxifen-treated breast cancer patients. Several studies have investigated the relationship between the gene encoding cytochrome P450 2D6 (CYP2D6) and tamoxifen treatment outcome, with widely heterogeneous results [4,5,8–10].

It has been suggested that CYP2D6 inhibitor use, aromatase inhibitor use and chemotherapy may account for some of these discrepancies [5]. Therefore, we examined the association between CYP2D6 genotype and early breast cancer events in tamoxifen-treated breast cancer patients, in relation to CYP2D6 inhibitor use, aromatase inhibitor use, and chemotherapy in a prospective cohort of tamoxifen-treated breast cancer patients in Sweden.

Material and methods

The design of the study has been described in detail elsewhere [11]. In brief, preoperative questionnaires on lifestyle and concomitant medications were completed by 634 breast cancer patients in Sweden between 2002 and 2008, among whom 333 patients had ER-positive tumors and received tamoxifen (Figure 1). Follow-up questionnaires were completed up to nine years postoperatively. Information on clinical data, breast cancer events, and tumor characteristics was obtained from patients’ charts, population registries, the Regional Tumor Registry, and pathology reports. ER and progesterone receptor (PR) status were obtained from each patient's pathology report. ER and PR status were determined by immunohistochemistry using the Dako LSAB kit system (Dako, Glostrup, Denmark) and the antibodies M7047 (ER) and M3569 (PR) (Dako). Tumors with more than 10% positive nuclear staining were considered ER-positive or PR-positive [12]. All tumors were analyzed at the Department of Pathology at Skåne University Hospital in Lund. Written informed consent was obtained from all patients. The study was approved by the Ethics Committee of the Lund University (Dnr 75-02 and 37-08).

Figure 1. Flow chart of the selection process of patients.

CYP2D6*3,*4,*6,*10, and *41 were analyzed using Genomic deoxyribonucleic acid (DNA) extracted from the leukocyte portion of whole blood. The SNPs were genotyped with custom TaqMan^® assay (CYP2D6*3), TaqMan^® Drug Metabolism Genotyping Assays (CYP2D6*4,*6,*10), and iPLEX reagents (CYP2D6*41) at Region Skåne Competence Centre (RSKC Malmö), Malmö University Hospital, Malmö, Sweden, using methods previously described [11]. Over 10% of the samples were run in duplicate with a concordance of 100%. CYP2D6 inhibiting drugs were classified in accordance with Flockhart et al. [13]. Only the use of the strongest inhibitor was considered. Patients with ER-negative tumors who had received tamoxifen treatment were excluded. Each patient received a CYP2D6 genotype activity score and a CYP2D6 activity score [14,15], and was then categorized as poor (PM), intermediate (IM), and extensive metabolizer (EM) (activity scores 0, 0.5–1.5, 2, respectively) [14].

Breast cancer-free survival was calculated from the pre-operative visit to the last follow-up visit or a non-breast cancer-related death or to a diagnosis of a breast cancer event prior to January 1, 2012. Survival analyses were performed using Kaplan-Meier and Cox regression models. Patients who had received pre-operative treatment (n = 17), patients with carcinoma in situ (n = 1), patients who lacked CYP2D6 genotype information (n = 7), and patients who both received pre-operative treatment and lacked CYP2D6 genotype information (n = 1) were excluded from the survival analyses. Survival in relation to combined tamoxifen and CYP2D6 inhibitor use was assessed though landmark analyses (landmark set as treatment initiation of tamoxifen and CYP2D6 inhibitors within nine months of study entry). A p-value of < 0.05 was taken to be significant. All p-values were two-sided.

Results

Patient characteristics for all 634 patients, and for the 333 tamoxifen-treated patients with ER-positive tumors, are presented in Table I. The median follow-up was 4.9 years. Frequency of the CYP2D6 genotypes, with and without stratification according to CYP2D6 genotype activity score and CYP2D6 activity score, is presented in Table II. Among the 307 tamoxifen-treated patients with invasive ER-positive tumors and CYP2D6 genotype information, 31 breast cancer events were observed. There was no association between early breast cancer events and CYP2D6 genotype metabolizer category (Figure 2a). Excluding chemotherapy-treated patients (Figure 2b) and patients who had received either chemotherapy and/or aromatase inhibitors (Figure 2c) generated similar results. Neither PM, nor IM were associated with early breast cancer events in a multivariable model compared to EM [adjusted hazard ratio (HR_PM) 0.50; 95% CI 0.07–3.82; p = 0.50], [adjusted (HR_IM) 1.00; 95% CI 0.47–2.11; p = 1.00], adjusting for invasive tumor size (> 20 mm, or muscular or skin involvement), any axillary lymph node involvement, age at diagnosis, and histological grade III.

Figure 2. Kaplan-Meier estimate of breast cancer-free survival in relation to CYP2D6 genotype activity score. a) among tamoxifen-treated patients with invasive ER-positive tumors (LogRank, p_trend = 0.48). Adjusted HR_PM (0.50; 95% CI 0.07–3.82; p = 0.50) and adjusted HR_IM (1.00; 95% CI 0.47–2.11; p = 1.00) compared with EMs. b) among tamoxifen-treated patients with invasive ER-positive tumors who had not received chemotherapy (LogRank, p_trend = 0.70). c) among tamoxifen-treated patients with invasive ER-positive tumors who had not received chemotherapy and/or aromatase inhibitors (LogRank, p_trend = 0.49).

Table I. Patient characteristics for all 634 patients, and for the 333 tamoxifen-treated patients with ER-positive tumors (in gray).

Table II. Frequency of the CYP2D6 genotypes in all patients (n = 634), and for the 333 tamoxifen-treated patients with ER-positive tumors (in gray) with and without stratification according to CYP2D6 genotype activity score and CYP2D6 activity score. CYP2D6 genotype was missing for 18 of the 634 patients.

Among the 301 genotyped tamoxifen-treated patients with invasive ER-positive tumors who had started tamoxifen treatment within nine months of study entry, 11 also reported use of strong CYP2D6 inhibitors, and 11 reported use of intermediate CYP2D6 inhibitors within nine months of study entry. Six of the tamoxifen-treated patients commenced tamoxifen after nine months, none of which had a breast cancer event. Two patients commenced strong CYP2D6 inhibitors use, and three patients commenced intermediate CYP2D6 inhibitors use after nine months but before five years past study entry; none of these five patients had a breast cancer event.

In the landmark analyses, CYP2D6 activity score (which takes both genotype and CYP2D6 inhibitor use into account) was not associated with early breast cancer events in the univariable (LogRank, p_trend = 0.44) or in the multivariable model. Tamoxifen treatment duration was not associated with CYP2D6 genotype, age, aromatase inhibitor or chemotherapy treatment.

Discussion

In this prospective cohort of tamoxifen-treated breast cancer patients in Sweden, there was no evidence that poor CYP2D6 metabolizers had an impaired tamoxifen treatment outcome. In accordance with several previous studies of breast cancer patients in Scandinavia [10,16,17], neither CYP2D6 genotype nor CYP2D6 inhibitor use was associated with early events.

The present study has several strengths. It is population-based [11] and uses Swedish population registries for optimal follow-up. All polymorphisms were analyzed from germline DNA, and multiple polymorphisms were included. Also, blood for genotyping was collected at study entry, and CYP2D6 inhibitor use was collected for the entire cohort of 634 patients. A total number of 38 patients (6%) used either strong or intermediate CYP2D6 inhibitors, which is comparable to a previous publication [18]. The main limitation of the study is the sample size. The number of tamoxifen monotherapy-treated patients was relatively small, but the lack of association between CYP2D6 genotype and disease-free survival seen in the present study has also been reported from previous studies conducted in larger cohorts [5,19].

CYP2D6 inhibitor use, chemotherapy, or aromatase inhibitor therapy did not account for the absence of an association between CYP2D6 genotype and tamoxifen treatment outcome in this study. Many antidepressants are metabolized by enzymes other than CYP2D6 that are involved in tamoxifen metabolism, such as CYP2C19 [20] and CYP2C8/9 [6,21]. This could in part explain the association between antidepressant use and breast cancer-free survival found in previous studies [6,22]. Other genetic variants [10,23,24], and lifestyle factors such as a high coffee intake, [25] have been associated with survival among tamoxifen-treated breast cancer patients in Sweden. CYP2D6 may be of minor importance for prediction of tamoxifen response in Scandinavia [9,10,16,17].

Acknowledgments

We wish to thank our research nurses: Anette Ahlin Gullers, Monika Meszaros, Maj-Britt Hedenblad, Karin Henriksson, Anette Möller, and Linda Ågren. We thank Sol-Britt Olsson, Nils-Gunnar Lundin, and Kristina Lövgren for taking care of blood samples. We also wish to thank Erika Bågeman, Maria Simonsson, and Maria Henningson for taking care of blood samples and data entry, and Dr Eric Dryver for proofreading.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by grants from The Swedish Cancer Society CAN 2011/497, the Medical Research Council K2012-54X-22027-01-3 (PI H Jernström), the Medical Faculty at Lund University, the Mrs. Berta Kamprad Foundation, the Gunnar Nilsson Foundation, the Swedish Breast Cancer Group (BRO), the South Swedish Health Care Region (Region Skåne ALF), Konung Gustaf V:s Jubileumsfond, and the Lund Hospital Fund.