Abstract
Background: The transport protein P-glycoprotein, which is encoded by the multidrug-resistance ABCB1 gene, is crucially involved in the export of taxanes and other cytotoxic substances out of the cell. Treatment response to paclitaxel has been shown to correlate with ABCB1 gene polymorphisms. Data regarding the prognostic value of ABCB1 gene polymorphisms in ovarian cancer patients is conflicting. Materials and Methods: The present study evaluates the association of two common ABCB1 gene polymorphisms, namely G2677T/A in exon 21 (rs2032582) and C3435T in exon 26 (rs1045642), and survival in 106 Caucasian women with ovarian cancer. Results: The two ABCB1 gene polymorphisms (G2677T/A and C3435T) were associated neither with disease-free (p=0.8 and p=0.9, respectively) nor with overall survival (p=0.9 and p=0.9, respectively). Tumor stage (p=0.01; p=0.01) and residual tumor mass (p=0.005; p=0.01), but not tumor grade and age at diagnosis were associated with disease-free and overall survival, respectively, in a multivariate analysis. Haplotype analysis did not reveal any association between the combined effect of the two gene polymorphisms and survival. Conclusion: In the present study, ABCB1 G2677T/A and ABCB1 C3435T gene polymorphisms were not found to be associated with prognosis in Caucasian women with ovarian cancer.
P-Glycoprotein (Pgp), which is encoded by the ATP-binding cassette subfamily B member 1 gene (ABCB1, often known as MDR1), is a 170 kDa transmembranous protein. It functions as an ATP-dependent efflux pump, transporting exogenous and endogenous substrates from the inside of the cell to the outside (1). Pgp is expressed in both nonmalignant tissue, such as the intestine and the blood–brain barrier, and malignant tissue, such as breast cancer and ovarian cancer tissue (2-4).
Numerous in vitro studies demonstrated that high expression of Pgp caused an elevated cellular efflux of xenobiotics (5, 6). Due to the elevated cellular clearance, high Pgp expression has been reported to be associated with a poor response to chemotherapeutic agents (7, 8), whereas tissue with low functional Pgp is more sensitive to various chemotherapeutic agents (9). Moreover, high Pgp expression is thought to lead to a multidrug-resistant phenotype (3, 4).
High Pgp expression is caused by elevated mRNA expression of the ABCB1 gene. ABCB1 expression seems to be higher in non-responders to paclitaxel/carboplatin compared to responders (10, 11), and has been reported to be a useful predictor of treatment response to taxane-containing chemotherapy (11). The ABCB1 gene is located on chromosome 7q21.1 in humans (5). Various single nucleotide polymorphisms (SNPs) have been described in Caucasian women; the most interesting SNPs are ABCB1 G2677T/A in exon 21 (rs2032582) and ABCB1 C3435T in exon 26 (rs1045642), as they have been shown to correlate with Pgp expression (12, 13). The homozygous mutant genotype of the two investigated polymorphisms has been reported to lead to lower Pgp levels, causing a lower cellular clearance of chemotherapeutic substances such as paclitaxel (12, 14).
These two gene polymorphisms have been studied in various malignancies and are associated with the risk for endometrial (15), breast (16), gastric (17) and colon cancer (18), as well as leukemia (19). Moreover, the homozygous wild-type genotypes of ABCB1 G2677T/A and ABCB1 C3435T have been reported to be associated with a better response to preoperative chemotherapy and better prognosis in breast (20) and esophageal cancer (21), acute myeloid leukemia (13, 22) and multiple myeloma (23).
Data regarding the association of ABCB1 gene polymorphisms and prognosis in women with ovarian cancer are conflicting. One study comprising 914 women did not observe any association between ABCB1 G2677T/A anddisease-free survival (24, 25). In contrast, a recent study by an Australian study group comprising 309 women with epithelial ovarian cancer observed a prolonged disease-free survival in women carrying the T/A allele of the ABCB1 G2677T/A gene polymorphism (26). Another smaller study (n=53) also reported the T/A allele of the ABCB1 G2677T/A gene polymorphism to be associated with a prolonged disease-free interval (12).
Based on the prominent role of ABCB1 in predicting treatment response to chemotherapy, we evaluated the association between the two gene polymorphisms ABCB1 G2677T/A in exon 21 and ABCB1 C3435T in exon 26 and prognosis in Caucasian women with ovarian cancer.
Patients and Methods
Patients. In the present study, we included 106 Caucasian women with ovarian cancer. Patients were included and treated at the Department of Gynecology and Gynecologic Oncology, Medical University of Vienna, Vienna, Austria. Approval for this study was obtained by the Institutional Review Board. Patients were treated according to standards of the respective institution with upfront surgery and adjuvant platinum-based chemotherapy. Surgical staging according to FIGO (International Federation of Gynecologists and Obstetricians) guidelines was performed, including hysterectomy, bilateral salpingo-oophorectomy, pelvic and/or para-aortic lymphadenectomy, appendectomy, omentectomy and cytoreductive procedure in order to resect all gross tumor. All patients with tumor stages FIGO Ic to III and all patients with clear cell carcinoma received a platinum-based chemotherapy. Patients wishing to preserve fertility and those with tumor stage FIGO Ia were treated with conservative surgery (unilateral salpingo-oophorectomy) and full surgical staging including washings, omentectomy, appendectomy, node biopsies and a thorough abdominal exploration with biopsies of all suspicious areas. Post-therapeutically, all patients were followed up four times annually, including pelvic examination, abdominal ultrasound examination, and serum tumor marker evaluation. Progression-free survival was defined as the time between date of histological diagnosis and the first sign of disease recurrence based on the Response Evaluation Criteria in solid Tumors (RECIST).
DNA extraction. DNA was obtained from peripheral venous blood using a commercially available DNA extraction kit (DNA Extraction System I; ViennaLab, Vienna, Austria) before therapy (surgery or neoadjuvant chemotherapy) was started. The extracted DNA was stored at 4°C until analyzed.
Pyrosequencing. Primer pairs 5′-GCAGGAGTTGTTGAAATG-3′ and 5′-TTAGTTTGACTCACCTTCC-3′, and 5′-CCTATGGA GACAACAGCC-3′ and 5′-GAGAGACTTACATTAGGCAG-3′ were used to amplify 89 bp and 106 bp fragments of the ABCB1 gene (mRNA: NM_000927, DNA: http://genome.ucsc.edu/), respectively. Polymerase chain reaction was carried out in a total volume of 25 μl, including 25 ng template, 5 pmol of each sense and antisense primers, and puReTaq Ready-To-Go PCR Beads (Amersham Biosciences, UK), which contain 2.5 units of puReTaq DNA polymerase, 10 mM Tris-HCl (pH 9.0 at room temperature), 50 mM KCl, 1.5m M MgCl2, 200 μM dATP, dCTP, dGTP and dTTP, and stabilizers including bovine serum albumin. The reaction was performed on a Perkin-Elmer GeneAmp PCR system 9600 (PerkinElmer, USA) with a primary denaturation step at 94°C for 1 minute, 40 cycles at 94°C for 30 seconds, at 50°C for 30 seconds, and at 72°C for 30 seconds. This was followed by a final incubation at 72°C for 7 minutes.
Patients' characteristics.
The polymorphism was detected using Pyrosequencer PSQ 96 and PSQ 96 SNP Reagent Kit using 25 μl PCR product according to the manufacturer's instructions; 5 pmol of the sequencing primers 5′-AGAAAGAACTAGAAGG-3′ and 5′-GTCACAGGAAGAGAT-3′ were applied to detect the polymorphisms in exon 21 and 26, respectively.
Statistics. After testing for normality using Kolmogorov-Smirnov test, continuous parameters are given as means [standard deviation (SD)]. Parameters were compared using Student's t-tests or Chi-square tests where appropriate. Results are given as p-values with odds ratio (OR) and 95% confidence intervals (CI). Survival probabilities were calculated by the product limit method of Kaplan and Meier. Differences between groups were tested using the log-rank test. The results were analyzed for the endpoint of disease-free and overall survival. Patients disease-free or still alive at the time of last follow-up were censored at the last follow-up date. Survival time was measured from the date of diagnosis. A multivariate Cox regression model was performed for disease-free survival and overall survival comprising tumor stage, tumor grade, residual tumor mass, patient's age at diagnosis, and the two ABCB1 gene polymorphisms. Haplotype frequencies were calculated using SAS/Genetics software. Association between haplotypes and ovarian cancer survival were calculated using a Cox regression model treating each haplotype as an independent continuous variable.
Univariate Kaplan-Meier analysis and multivariate Cox regression model of prognostic covariates in women with ovarian cancer.
Association between ABCB1 haplotypes and overall survival in women with ovarian cancer.
SAS System software (Version 9.1 SAS Institute Inc., Cary, NC, USA) and SPSS (SPSS 11.0, SPSS Inc. Chicago, IL, USA) were used for statistical analysis. P-values less than 0.05 were considered statistically significant.
Results
Patients' characteristics are shown in Table I. Genotype frequencies in patients with ovarian cancer were as follows: ABCB1 G2677T/A 42.9% (G/G), 44.8% (G/T), and 12.4% (T/T), and ABCB1 C3435T 22.6% (C/C), 60.4% (C/T), and 17.0% (T/T). The distributions of the genotype frequencies for ABCB1 G2677T/A and C3435T in women with ovarian cancer were in Hardy-Weinberg equilibrium (p=0.9 and p=0.2, respectively). No associations were found between ABCB1 G2677T/A and C3435T and clinicopathological parameters, i.e. tumor stage (p=0.7 and p=0.6), tumor grade (p=0.4 and p=0.8), residual tumor mass (p=0.1 and p=0.5), and age at diagnosis (p=0.9 and p=0.8), respectively.
In a univariate Kaplan-Meier analysis, tumor stage, tumor grade, residual tumor mass, and patient's age at diagnosis, but neither ABCB1 G2677T/A nor ABCB1 C3435T gene polymorphisms were associated with disease-free and overall survival (Table II). In a multivariate Cox regression model, tumor stage and tumor residual mass, but not tumor grade, patient's age at diagnosis, ABCB1 G2677T/A, nor ABCB1 C3435T gene polymorphisms, were associated with disease-free and overall survival (Table II). The combined prognostic effect of the two gene polymorphisms was investigated by haplotype analysis (Table III). No significant associations between haplotypes and disease-free or overall survival were observed.
Discussion
The ABCB1 gene polymorphisms seem to have a certain predictive value for the treatment response in women with ovarian cancer (11, 12, 20). We investigated whether two important ABCB1 gene polymorphisms are associated with prognosis of women with epithelial ovarian cancer.
Although the ABCB1 pathway is thought to play a crucial role in treatment response and drug resistance in women with ovarian cancer (1, 9, 11, 12), our findings do not support the hypothesis that the investigated ABCB1 gene polymorphisms, whether combined or as independent factors, have a prognostic value in women with ovarian cancer, nor do they appear to be associated with the biological phenotype. Our negative finding is in line with the largest study investigating the role of ABCB1 G2677T/A gene polymorphism and prognosis of women with ovarian cancer (24). It is in contrast to a study performed by an Australian study group comprising 309 patients (26). Of note, the latter study observed an association between ABCB1 G2677T/A gene polymorphism and disease-free survival, but not overall survival, only in a subset of optimally debulked patients (26). The different findings compared to the Australian study might be caused by slightly diverging genotype distribution. They observed a higher number of heterozygous and homozygous mutant ABCB1 G2677T/A genotypes, which were seen as a risk factor for shortened progression-free survival, than we did (69.7% vs. 57.5%) (26).
Clinical drug resistance is almost certainly multifactorial (27). Recently, various pathways have been identified and shown to be associated with drug resistance and a subsequent poorer prognosis in women with ovarian cancer (28-30). Different molecular mechanisms might lead to different types of drug resistance (27). Furthermore, our sample size might be too small to demonstrate the independent effect of ABCB1 G2677T/A and ABCB1 C3435T gene polymorphisms on survival in women with ovarian cancer or perform a subanalysis to evaluate only optimally debulked patients. Of note, the aim of our study was to investigate the prognostic but not the predictive value, regarding response to chemotherapy, of ABCB1 gene polymorphisms.
In conclusion, we were not able to observe any association between ABCB1 G2677T/A and ABCB1 C3435T gene polymorphisms and prognosis in Caucasian women with epithelial ovarian cancer.
Footnotes
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Financial Support
Ludwig Boltzmann-Institute of Gynecology and Gynecologic Oncology, Vienna, Austria.
- Received October 8, 2009.
- Revision received April 12, 2010.
- Accepted April 22, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
