ABCB1 polymorphism as prognostic factor in breast cancer patients treated with docetaxel and doxorubicin neoadjuvant chemotherapy

Expression of the adenosine triphosphate-binding cassette B1 (ABCB1) transporter and P-glycoprotein are associated with resistance to anticancer drugs. The purpose of this study was to investigate the role of single nucleotide polymorphism in the ABCB1 and CYP3A genes in breast cancer patients who were treated with neoadjuvant chemotherapy. Stage II/III breast cancer patients were treated with three cycles of neoadjuvant, after which the patients received curative surgery and adjuvant chemotherapy. The polymorphisms of ABCB1 and CYP3A were genotyped. The correlation of polymorphism of ABCB1, CYP3A, and clinical outcomes was analyzed. Among the 216 patients, ABCB1 3435TT genotype had a longer overall survival (OS). than CC/CT. Multivariate analyses demonstrated that good PS, invasive ductal carcinoma, non-triple negative phenotype and initial operable stage were significantly associated with a lower death risk. ABCB1 3435TT genotype had a higher AUC than CC/CT for docetaxel. These higher AUCs in the C3435TT was associated with increased toxicities of neutropenia and diarrhea. This study showed that the genetic polymorphism of ABCB1 C3435T might be associated with a longer OS. Our results also suggest that the prediction of docetaxel toxicity might be possible for C3435T polymorphism. This study results provides valuable information on individualized therapy according to genotypes.

Expression of the adenosine triphosphate-binding cassette B1 (ABCB1) transporter and P-glycoprotein are associated with resistance to anticancer drugs. The purpose of this study was to investigate the role of single nucleotide polymorphism in the ABCB1 and CYP3A genes in breast cancer patients who were treated with neoadjuvant chemotherapy. Stage II ⁄ III breast cancer patients were treated with three cycles of neoadjuvant, after which the patients received curative surgery and adjuvant chemotherapy. The polymorphisms of ABCB1 and CYP3A were genotyped. The correlation of polymorphism of ABCB1, CYP3A, and clinical outcomes was analyzed. Among the 216 patients, ABCB1 3435TT genotype had a longer overall survival (OS). than CC ⁄ CT. Multivariate analyses demonstrated that good PS, invasive ductal carcinoma, non-triple negative phenotype and initial operable stage were significantly associated with a lower death risk. ABCB1 3435TT genotype had a higher AUC than CC ⁄ CT for docetaxel. These higher AUCs in the C3435TT was associated with increased toxicities of neutropenia and diarrhea. This study showed that the genetic polymorphism of ABCB1 C3435T might be associated with a longer OS. Our results also suggest that the prediction of docetaxel toxicity might be possible for C3435T polymorphism. This study results provides valuable information on individualized therapy according to genotypes. R andomized trials have proven the safety of neoadjuvant chemotherapy with comparable efficacy to adjuvant chemotherapy. (1,2) These studies provide the theoretical roles supporting neoadjuvant chemotherapy. First of all, "downstaging" of the tumor from inoperable to operable stage in select patients may allow a substantial proportion of women to benefit from conservative surgery instead of having to resort to more radical approaches. (3) Second, post-surgical treatment strategy could be optimized based on the previous tumor response during neoadjuvant chemotherapy. Last but not least, a subset of patients could achieve pathologic complete remission (pCR) from neoadjuvant therapy, which has been shown to be associated with a good prognosis. (4,5) As neoadjuvant chemotherapy is becoming increasingly more important in clinical practice, a disputed issue is the development of resistance to the cytotoxic agents as well as cross-resistance to substances patients have never been exposed to before. Inter-individual variability of pharmacokinetics of anti-cancer drugs constitutes a major limitation to their clinical use. Since a variety of commonly used chemotherapeutic agents such as docetaxel and doxorubicin have narrow therapeutic indices and significant inter-individual variations in drug disposition, great efforts have been put in determining the mechanism of person-to-person pharmacokinetic diversities.
Single nucleotide polymorphisms (SNPs) in adenosine triphosphate-binding cassette B1 (ABCB1) and cytochrome P450 (CYP) genes are among potential candidates that explain the inter-individual variations in drug disposition. SNPs in ABCB1 gene are associated with phenotypic variation in P-glycoproteins (P-gp), a membrane-bound efflux pump, which removes chemotherapeutic drugs including docetaxel and doxorubicin from the cells. The intestinal P-gp plays a main role in the fecal elimination of drugs by modulating reabsorption of the drug after hepatobiliary secretion. (6) The percentage of tumors expressing P-gp in breast cancer was 41.2% and the expression rate of P-gp increased after treatment which was three times more likely to fail in chemotherapy. (7) Recently, SNPs of the ABCB1 gene have been reported to be associated with taxane clearance and the clinical outcome of patients who were treated with taxane. (8,9) Doxorubicin was also shown to be functionally affected by SNPs of ABCB1 with regards to the pharmacokinetics. (10,11) It has been shown that ABCB1 C3435T polymorphism in exon 26 is significantly related to clinical toxicity and high blood levels of docetaxel. (12) ABCB1 G2677T ⁄ A polymorphism in exon 21 are associated with treatment outcomes after paclitaxel monotherapy. (11) ABCB1 C1236T polymorphism in exon 12 is associated with docetaxel clearance. (8,9) Most chemotherapeutic agents are eliminated via hepatic metabolism, which is mediated by cytochrome P450 (CYP), mainly CYP 3A4 and CYP3A5. (13) Metabolism of docetaxel consists of CYP3A-mediated oxidation of the tert-butyl propionate side chain, which results in the formation of four metabolites with reduced cytotoxic activity. (14) CYP3A4*1B and CYP3A5*3 are common polymorphisms, which have been shown to have clinical implications in some studies. (14,15) CYP3A4*1B, an A-392G transition in the 5 0 -flanking region, is a promoter polymorphism. While the polymorphism is commonly found in the African-American population, (14) its minor allele frequency is estimated 0% in Chinese, Taiwanese, Chinese Americans, and Japanese Americans. (16,17) On the other hand, the CYP3A5*3 splice site variant that causes loss of hepatic expression of CYP3A5 is common, but does not account for inter-individual variability of docetaxel disposition in Asian populations. (18,19) P-gp may affect the extent of CYP3A-mediated metabolism of drugs by limiting the intracellular substrate availability. (20) This study hypothesized from the above findings that a genetic variant of ABCB1 and CYP3A5 contributes to the pharmacokinetic variability of docetaxel and doxorubicin.
This study investigated the distribution of SNPs in ABCB1 and CYP3A genes in breast cancer patients who were treated with neoadjuvant docetaxel and doxorubicin chemotherapy. The correlation between the genetic polymorphism, plasma concentration of chemotherapeutic agents and clinical outcomes was also explored.

Materials and Methods
Patients and study design. From September 2003 to September 2008, pathologically proven, previously untreated, clinical stage II or III breast cancer patients were enrolled in this prospective study. Patients had to have an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0-2, objective measurable disease, and adequate bone marrow, hepatic, cardiac, and renal functions. Initial evaluations included physical examination, mammography, breast ultrasonography, computed tomography (CT) of the chest, bone scan, and breast magnetic resonance imaging (MRI). Initial nodal staging was evaluated by the physical examination and CT.
All patients were treated with three cycles of neoadjuvant chemotherapy which consisted of docetaxel (75 mg ⁄ m 2 ) and doxorubicin (60 mg ⁄ m 2 ) by intravenous infusion. (21) Granulocyte colony-stimulating factor as primary prophylaxis was injected for 5 days every cycle. After completion of neoadjuvant treatment, the patients were re-evaluated for the response. After the primary surgery, patients were treated with three more cycles of docetaxel and doxorubicin as the adjuvant chemotherapy. (21,22) Adequate radiation or hormonal therapy was performed as indicated. (23,24) The radiological response was evaluated using breast MRI for the primary breast lesion and chest CT for the lymph node lesions with RECIST criteria 1.0. (25) Pathologic complete response (pCR) was defined as complete disappearance of invasive carcinoma in both breast and axillary lymph nodes after three cycles of chemotherapy. For estrogen receptor (ER) and progesterone receptor (PR), cases with 10% or more positive staining were grouped as positive. Human epidermal growth factor receptor-2 (HER2) overexpression was demonstrated by immunohistochemistry scoring or fluorescence in situ hybridization (FISH) in paraffin Analysis of ABCB1 and CYP3A genomic polymorphism. Whole blood samples were obtained before the neoadjuvant chemotherapy. Genomic DNA was isolated from mononuclear cells of peripheral blood cells using QiaAmp blood mini kit (Qiagen, Hilden, Germany). ABCB1 C3435T, (26,27) G2677T ⁄ A (11,27) and C1236T (28) polymorphisms were genotyped by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) assays as previously described. (29) CYP3A4*1B and CYP3A5*3 polymorphisms were done by the GoldenGate assay (Illumina, San Diego, CA, USA) that consisted of 96 custom selected SNPs within 34 genes. The dissolved biotinylated DNA pellet was extended and ligated. The supernatant was then used in PCR amplification. Double-stranded PCR products were immobilized onto paramagnetic particles, B Reagent (Illumina). The bound PCR products were denatured and the released ssDNAs were tralized with hybridization reagent (Illumina). Hybridization was done and then imaged using a BeadArray Reader (Illumina).
Pharmacokinetic studies. Seventy-two out of 216 patients participated in the prospective pharmacokinetic study of docetaxel and doxorubicin. These patients were grouped as the pharmacokinetic (PK) study group. Pharmacokinetic samplings were done at three time points. The plasma concentrations of docetaxel and doxorubicin at pre-concentration (just before infusion), post-concentration (just after infusion), and 24 h-concentration after infusion were determined by liquid chromatography tandem mass (LC ⁄ MS ⁄ MS) assays. Mass spectrometric analysis was done using an API 3000 MS system (Applied Biosystems, Foster City, CA, USA). All plasma samples were subjected to the sample preparation procedure described previously. (30) With the above three blood samples, a graph was plotted of the sampling point as the x-axis against the drug concentration as the y-axis and was able to draw a diagram corresponding to the area under the plasma concentration (AUC) until 24 h after drug infusion. Based on this diagram, the drug concentration was calculated and was defined as the AUC in this study. The AUC of docetaxel and doxorubicin were dictated as AUC doc , and AUC dox, respectively. The differences between the AUCs were examined until the 24 h-concentration after infusion, according to the genotypes of ABCB1 and CYP3A5.
Statistical analysis. This is a project of a phase II trial. The primary endpoint in this study was pCR. Secondary end points included rate of breast conserving operation, toxicity, relapse-free survival, overall survival and predictive ⁄ prognostic factors. The association between the allelic frequency of ABCB1 and clinical outcomes was assessed by the v 2 test or Fisher's exact test. The RFS and OS were estimated by the Kaplan-Meier method, and the difference in survival was compared using the log-rank test. Cox regression model was used to compare the clinical outcomes among the  genotypes of the ABCB1 gene and CYP3A5. The crude hazard ratio (HR) of clinical events and the corresponding 95% confidence intervals (CI) were estimated. For genotype-pharmacokinetic study, Mann-Whitney test or Kruskal-Wallis test was done to compare the AUCs between post-concentration (just after infusion) and 24 h-concentration after infusion in the plasma concentrations according to the genotypes. Odds ratios and their respective 95% confidence intervals were used to show the relationship of the toxicities and covariates including the ABCB1 gene. SPSS program (version 12.0, Chicago, IL, USA) was used for all analyses, and two-sided P-value <0.05 was considered statistically significant.

Results
Patients and treatment outcomes. A total of 216 breast cancer patients were enrolled. The median follow-up duration was 85.4 months (range 26.2-117.8 months) and 75 patients (34.7%) experienced relapse and 43 patients (19.9%) died. Baseline characteristics of the study population are presented in Table 1. The median age of the study population was 44 years (range 25-69 years). ABCB1 C3435T and ABCB1 C1236T were divided into two groups as the CC ⁄ CT vs. TT genotype and the ABCB1 G2677T ⁄ A was grouped into the GG vs. Non-GG group.
Pharmacokinetic analysis was done in 72 patients. Baseline characteristics of these patients did not differ greatly from those of the total patient group.
Association between genotypes and treatment outcomes. Frequency of ABCB1 and CYP3A genotypes. The frequency of ABCB1 3435TT genotype was 14.4% in total 216 patients and 9.7% in the PK group of 72 patients. In PK group, the AA genotype of CYP3A4 was 100% and the AA genotype of CYP3A5 had the lowest percentage, 4.2%. The proportion of each genotype was maintained similarly in 72 PK group patients. Each observed genotype distribution of ABCB1 genes conformed to Hardy-Weinberg equilibrium (each P > 0.050). Linkage disequilibrium for all pairs of exons 26, 21, and 12 were observed by use of the PL-EM algorithm (P < 0.001).
Analysis of clinical outcomes of the total patients. The overall clinical response rate (RR) was 76.8%. Twenty patients (9.3%) achieved clinical complete response (cCR) and 18 (8.3%) achieved pCR. Among the PK group, RR was 82.0% with 4.2% of pCR. ABCB1 C3435T, G2677T ⁄ A, and C1236T SNPs did not predict the responders. As shown in Figure 1, ABCB1 3435TT genotype had a longer OS than the CT ⁄ TT genotype with statistical significance (P = 0.024). A similar trend was observed for the RFS, that is, ABCB1 3435TT genotype tended to have a longer RFS although it was not statistically significant (P = 0.234). ABCB1 G2677T ⁄ A and C1236T were analyzed, and there were no significant differences in the RFS and OS (Fig. S1).
With univariate analysis of the OS, good PS (ECOG 0-1), invasive ductal carcinoma, initial operable stage, ER-positivity, non-triple negative phenotype, breast conserving surgery and the TT genotype of ABCB1 C3435T were associated with a lower risk of death (Table 2)   associated with the OS ( Table 3). The ABCB1 3435TT genotype was also associated with a lower risk of death with marginal significance not shown to be an independent prognostic factor (HR 0.245, 95% CI = 0.059-1.026; P = 0.054). The result was similar when breast cancer staging was adjusted instead of operable staging (HR 0.238; 95% CI, 0.057-0.999; P = 0.050).

Discussion
In recent years, various genetic variants in ABCB1 have been described to affect the transporter expression and function. (31) Docetaxel and doxorubicin are subjected to transport by the ABCB1 transporter. ABCB1 3435C (glutamate) to T (glutamate) substitution does not result in an amino acid sequence change nor is this gene located in a regulatory region. (8) This synonymous ABCB1 C3435T polymorphism has been associated with mRNA instability and a lower expression of P-gp in the duodenum. (27) P-gp expression and ABCB1 C3435T polymorphism have been identified as an independent factor for RFS and RR for chemotherapy in breast cancer. (11) Hoffmeyer et al. reported that the TT genotype of C3435T was correlated with reduced expression of P-gp and, therefore, reduced cellular elimination and maintained higher plasmaconcentrations of chemotherapeutic drugs. (27,32) From these results, we hypothesize that patients who have the ABCB1 3435TT genotype might show a better treatment response to docetaxel and doxorubicin chemotherapy and get survival benefit compared to ABCB1 3435 CC ⁄ CT.
This study was designed to identify the clinical impact of ABCB1 SNPs in Korean patients with breast cancer who were treated with docetaxel and doxorubicin, a regimen widely accepted as neoadjuvant chemotherapeutic agents. (21) In accordance with previous studies, this study showed that the TT genotype of ABCB1 3435 was associated with a longer OS and higher plasma concentration of docetaxel than CT ⁄ TT. (33,34) It also suggests that a higher plasma level of docetaxel was maintained for a longer time in the TT genotype of ABCB1 translating into more intense exposure to docetaxel. As hematopoietic stem cells and intestinal epithelial cells are reported to express P-gp, (35) higher drug concentrations in homozygote 3435TT leads to more frequent grade 3-4 neutropenia, febrile neutropenia, and diarrhea. Since the number of patients in the pharmacokinetic group was small, the OS tended to be longer in the ABCB1 3435TT genotype without statistical significance. There was a trend toward a higher percentage of grade 3-4 neutropenia in the ABCB1 3435TT subjects in the PK group (28.6% versus about 3.1% for the CC ⁄ CT subjects). It provides valuable information to clinicians that the ABCB1 homozygote carriers have increased efficacy and increased toxicity simultaneously. There were no significant differences in the OS at the analyzed data of G2677T ⁄ A and C1236T. To our knowledge, this is the first study demonstrating that ABCB1 C3435T polymorphism is significantly associated with plasma docetaxel concentration and OS after neoadjuvant docetaxel ⁄ doxorubicin combination chemotherapy in Asian patients with stage II or III breast cancer.
Since liver metabolism of docetaxel to inactive hydroxylated metabolites is mediated by CYP3A4 and CYP3A5, (12,36) polymorphisms of such enzymes could affect the survival outcome of the treatment in breast cancer patients. Intuitively, it is reasonable to infer that the side effects of docetaxel treatment could be related to those metabolic enzymes. The most common variant, CYP3A4*1B allele founded in the African-American population, was not detected in the present study as expected based on previously published data. (37) In contrast to CYP3A4*1B, the frequency of CYP3A5*3 allele was 65-85% in Asia (18) and 76.8% in our study. The CYP3A5 is linked to a common transition in intron 3 of the CYP3A5 gene (CYP3A5 *3) which introduces a frameshift during translation and results in a truncated, nonfunctional protein. (38) At this point, homozygote (GG genotype,*3 ⁄ *3) for CYP3A5*3 allele would be expected to be also strongly associated with low CYP3A5 protein content and AUC in a homozygote for the CYP3A5*3 allele which would be higher than CYP3A5*1. However, our findings show somewhat conflicting results: the homozygote of CYP3A5*3 tend to have a lower docetaxel concentration and a higher risk of grade 3 diarrhea than the CYP3A5*1 ⁄ *1 genotype (P = 0.072). Concordantly, Goh et al. reported the same result that docetaxel clearance in patients with at least one CYP3A5*1 allele was significantly lower than CYP3A5*3 homozygotes. (19) What causes this difference in predictions is as follows: the AA genotype (CYP3A5*1 ⁄ *1) of CYP3A5 was only in three patients (4.2%) and was correlated with the TT genotype of C3435T with statistical significance. So a high docetaxel AUC revealed in the ABCB1 3435TT could be associated with the AA genotype of CYP3A5. Although CYP3A5 can account for >50% of the total CYP3A, we speculate that docetaxel metabolism was affected by the presence of the CYP3A5*3 allele to a small degree.
One of the merits of this study is the homogeneity of the population who received the same neoadjuvant chemotherapy followed by surgery for stage II ⁄ III breast cancer. Second, the study was designed prospectively; pharmacogenomics samples and plasma drug concentration samples were collected at specific time points according to the written prospective protocol in the chemotherapy-na€ ıve patients. The clinical data were gained through the same therapeutic scheme. With all of these advantages, the major limitation of this study is the relatively small sample size of the pharmacokinetic study population.
However, the distribution of genotype in this study was well matched to what has been reported previously in Koreans, and in a line with those with Asian population. (39) In a sense, we believe the small sample size of this study did not led to selection bias, and thus does not undermine the significance of the findings.
In conclusion, this study shows that the genetic polymorphism of ABCB1 C3435T is associated with higher docetaxel concentration, a longer OS and more frequent toxicities. Our results suggest the future of guided drug selection by predicting drug toxicities according to the patient's genotype. Further prospective trials with large scale as well as in vitro and in vivo functional studies are warranted.