Paclitaxel is commonly used in the treatment of breast cancer. Variability in paclitaxel clearance may contribute to the unpredictability of clinical outcomes. We assessed genomic DNA from the plasma of 93 patients with high-risk primary or stage IV breast cancer, who received dose-intense paclitaxel, doxorubicin and cyclophosphamide. Eight polymorphisms in six genes associated with metabolism and transport of paclitaxel were analyzed using Pyrosequencing. We found no association between ABCB1, ABCG2, CYP1B1, CYP3A4, CYP3A5 and CYP2C8 genotypes and paclitaxel clearance. However, patients homozygous for the CYP1B1*3 allele had a significantly longer progression-free survival than patients with at least one Valine allele (P=0.037). This finding could reflect altered paclitaxel metabolism, however, the finding was independent of paclitaxel clearance. Alternatively, the role of CYP1B1 in estrogen metabolism may influence the risk of invasive or paclitaxel resistant breast cancer in patients carrying the CYP1B1*3 allele.
Paclitaxel is one of the most active single agents in the treatment of breast cancer and is an important component of many commonly used combination regimens.1 Despite its clinical activity, variability in toxicity and response remain a major problem for patients receiving paclitaxel. Significant variability (4- to 10-fold) in paclitaxel clearance may contribute to the unpredictability of clinical outcomes.2 However, to date the contribution of genetic variation to these interindividual differences has not been defined clearly. In particular, there is a paucity of pharmacogenetic studies focused on paclitaxel therapy for breast cancer.
Paclitaxel elimination is regulated by a wide array of genes involved in metabolism and extracellular transport.3 Oxidative paclitaxel metabolism occurs via the cytochrome P450 pathway. Data from liver microsomes demonstrate that CYP2C8 and CYP3A4 are primarily responsible for paclitaxel metabolism,4, 5 with CYP2C8 demonstrating a 2.3-fold greater metabolite production than CYP3A4.5 The CY2C8*3 variant allele has been associated with decreased paclitaxel 6 α-hydroxylase activity in human cell lines and human liver microsomes.4, 6 Functional variants in the CYP3A gene family have been identified that could potentially impact drug metabolism. A polymorphism in the 5′ regulatory region of the gene, CYP3A4*1B, has been associated with significantly increased CYP3A4 transcriptional activity.7 A common polymorphism in CYP3A5, CYP3A5*3C, causes a splicing defect leading to significantly decreased CYP3A5 protein expression.8 The CYP3A5*3C allele encoding the truncated CYP3A5 protein is the most common allele in Caucasian populations (up to 95%).8 CYP1B1 is the most prominent human cytochrome in breast tissue9 and is often overexpressed in tumor cells.10, 11, 12 There is some evidence to suggest that the taxanes paclitaxel and docetaxel are competitive inhibitors of CYP1B1 activity13 and recent data has identified docetaxel as an effector of CYP1B1.14 The CYP1B1*3 polymorphism has been associated with increased CYP1B1 mRNA expression,15 increased catalytic activity16, 17, 18 and specifically altered estrogen hydroxylation activity with the potential to interfere with tumor biology.17, 19, 20, 21
Overexpression of the adenosine triphosphate-binding cassette transporters ABCB1 and ABCG2 has been associated with resistance to taxane-based agents in vitro,22 although there is minimal evidence to suggest a role for paclitaxel as a substrate for ABCG2. ABCB1 appears to be the main paclitaxel transporter23, 24 and variants in ABCB1 have been demonstrated to alter P-glycoprotein expression.25, 26 A non-synonymous variant in ABCG2 (421C>A; Q141K) has been associated with drug resistance in vitro and in vivo,27, 28, 29 and with survival in prostate cancer patients receiving docetaxel chemotherapy (P=0.05),30 consequently, despite the lack of evidence associating taxane transport with ABCG2 the 421C>A variant was included in this study.
We assessed polymorphisms in six genes associated with paclitaxel metabolism and transport to determine the contribution of inherited differences to the pharmacokinetics and progression-free survival of breast cancer patients receiving paclitaxel-containing therapy.
Results and discussion
Genotype and allele frequencies are shown in Table 1. When assessed separately by race the genotype frequencies did not deviate from Hardy–Weinberg equilibrium. Variant allele frequencies did not deviate significantly from those reported previously for predominantly Caucasian populations.4, 20, 27, 31, 32, 33
Using Kruskal–Wallis and analysis of variance (ANOVA) tests, we found no significant association between ABCB1, ABCG2, CYP1B1, CYP3A4, CYP3A5 and CYP2C8 genotypes and paclitaxel clearance (data not shown). However, χ2 analysis demonstrated that the CYP1B1*3 (4326 C>G; L432V) allele was significantly associated with progression-free survival (P=0.037). Patients homozygous for the Leucine allele (45%) had a significantly longer progression-free survival (median not yet reached) than patients with at least one Valine allele (median: 30 months). Kaplan–Meier analysis for progression-free survival demonstrated a significant association with CYP1B1*3 genotype (P=0.037; Figure 1). This significant association was independent of paclitaxel clearance, disease stage or estrogen/progesterone receptor status. No polymorphisms were significantly associated with overall survival (data not shown).
The lack of association between polymorphisms in paclitaxel metabolism and transport genes and paclitaxel clearance supports recent findings.34 One recent study in ovarian cancer identified an association between ABCB1 2677G>A/T and response to paclitaxel in 53 Swedish ovarian cancer patients,35 but this could not be replicated in a larger ovarian cancer study,36 and was not seen in the breast cancer patients in this study. Although in vitro evidence suggests a role for CYP2C8 and CYP3A4 in paclitaxel metabolism,4, 5 it is possible that other variables such as epigenetic regulation mask these effects in vivo. For example, the nuclear receptor, NR1I2 has been shown to regulate paclitaxel metabolism and efflux,37 and methylation status in ovarian tumor has been associated with response to taxane therapy.38 Alternatively, the multiple members of the paclitaxel drug elimination pathway may provide adequate redundancy to overcome genetic variation in any one gene.3, 39 The pharmacogenetics of paclitaxel may also be tumor-type dependent.
The present study involved patients receiving high-dose paclitaxel (575–775 mg/m2). It has been demonstrated that at the higher doses paclitaxel pharmacokinetics becomes non-linear and shows high inter-patient variability, possibly as a result of differences in elimination or saturable tissue distribution.2 Consequently, it is possible that novel pharmacogenetic associations previously not seen in patients receiving lower paclitaxel doses could have been identified here, and existing associations not replicated. In addition, cyclophosphamide and doxorubicin are also metabolized by CYP3A4 and CYP3A540, 41, 42 and this may in part mask any associations between polymorphisms in these genes and paclitaxel pharmacokinetics in the patients in this study. It is possible that paclitaxel in combination with different chemotherapy agents will have a different pharmacogenetic profile.
The significant association between CYP1B1*3 and progression-free survival could reflect altered paclitaxel metabolism. CYP1B1*3 is associated with increased estrogen hydroxylation activity,17 consequently, as a competitive substrate for CYP1B1,13 paclitaxel metabolism could be increased by the presence of the CYP1B1*3 alleles, thereby reducing the concentration of active paclitaxel. However, the suggestion that docetaxel is not metabolized by and yet binds to CYP1B1,14 thereby reducing available docetaxel in the cell, would be more supportive of a role for CYP1B1 in taxane efficacy without a direct effect on taxane metabolism. Alternatively, the alterations in CYP1B1 may influence tumor biology. CYP1B1*3 alleles are associated with biased formation of 4-hydroxyestradiol, which is potentially carcinogenic,18, 43 compared to formation of the lower risk 2-hydroxyestradiol metabolite.43 Consequently, the role of CYP1B1 in estrogen metabolism17, 19, 20 may influence the risk of invasive or paclitaxel resistant breast cancer in patients carrying the CYP1B1*3 allele. This study was performed in a small sample size and the data should be considered preliminary until validated in a large, prospective study.
Patients and methods
Genomic DNA was isolated from the plasma of 93 female patients (89% Caucasian; 8% Asian; 3% African-American, age range 30–63; median 48 years) with high-risk primary (84% stage IIIA through IIIC) or stage IV (16%) breast cancer. 74% of patients were estrogen/progesterone receptor positive. Patients received paclitaxel 575–775 mg/m2 infused over 24 h, doxorubicin 165 mg/m2 as a continuous infusion over 96 h, and cyclophosphamide 100 mg/kg, as consolidation therapy following either adjuvant chemotherapy (stages IIIA through IIIC) or after induction therapy (stage IV disease). Paclitaxel clearance was measured as described previously.2 Median overall follow-up of alive patients was 55 months. Appropriate written consent was obtained for all patients and this study was approved by the Institutional Review Board.
Eight polymorphisms in six genes associated with metabolism (CYP2C8*3 and *4; CYP1B1*3; CYP3A4*1B; CYP3A5*3C) and transport (ABCB1 2677 G>T/A and 1236 C>T; ABCG2 421 C>A) of paclitaxel were analyzed using Pyrosequencing technology as described previously (assay information available on request).28, 33, 44
Associations between genotype and paclitaxel clearance were performed using Kruskal–Wallis and ANOVA tests. Genotype-outcome associations were performed using χ2 and Kaplan–Meier analysis.
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We thank A Garsa for technical assistance with this project. This work was supported by CA 33572, CA 62505 (Duarte) and the Pharmacogenetics Research Network U01 GM63340 (St Louis).
Duality of interest
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