Polymorphism in glutathione S-transferase P 1 ( GSTP 1 ) on the effect of epirubicin or doxorubicin chemotherapy among breast cancer

To investigate the glutathione S-transferase P1 (GSTP1) expression with chemotherapy polymorphisms involved in the prognosis of breast cancer patients with epirubicin or doxorubicin chemotherapy among breast cancer. 219 primary breast cancer patients who were consecutively recruited in our study have been treated with anthracycline-based (epirubicin [E] or doxorubicin [A]) chemotherapy between March 2006 and March 2007. All the patients were followed up until January 2012. Genotyping was based upon duplex polymerase-chain-reaction with polymerase chain reaction with confronting two-pair primers (PCR-CTPP) method. The frequencies of GSTP polymorphisms were found have significant difference in progesterone receptor status (P<0.05). GSTP1 Val/Val genotype had significantly higher rates of response to chemotherapy when compared to GSTP1 Ile/Ile genotype, and the adjusted odds ratios (OR) (95% confidence interval, CI) of 2.19 (1.23 to 4.21). Additionally, the GSTP1 Val allele genotype had significantly higher rates of response to chemotherapy, with adjusted OR (95% CI) of 1.71 (1.12 to 2.76). Patients with glutathione S-transferase M1 (GSTM1) null genotype had a longer average survival time and significantly lower risk of death than those with non-null genotypes [Hazard ratio (HR) (95% CI) = 0.66 (0.31 to 0.93)]. Similarly, those carrying GSTP1 Val/Val genotype had 0.54-fold the risk of death of those with GSTP1 Ile/Ile [HR (95% CI) = 0.54(0.29 to 0.90)]. GSTP1 expression was found to be associated with response to chemotherapy treatment of breast cancer and Estrogen receptor (ER) status had some mechanism between GSTP1 polymorphism and response to chemotherapy.


INTRODUCTION
Breast cancer is by far the most frequent cancer among women with an estimated 1.38 million new cancer cases diagnosed in 2008 (23% of all cancers).In China, it is one of the most leading causes of death in Chinese women with an incidence of 14.2/10 5 (IARC, 2008).Neoadjuvant chemotherapy (NAC) is used to enhance the operability of breast cancer patients with advanced tumors who is known to enhance the operability of patients with advanced tumors previously considered inoperable, as *Corresponding author.E-mail: hejianmiao5666@163.com.
well as making breast-conserving surgery more feasible for patients for whom such surgery was previously not feasible due to large tumor size.Anthracycline-based chemotherapy regimens are preferred for downstaging breast cancer tumors (Bafaloukos, 2005).
Glutathione S-transferases (GST) are a multigene family of enzymes which catalyze the conjugation of glutathione to electrophilic xenobiotics to inactivate them, and thus prevent DNA damage and adduct formation (Watson et al., 1998).There were five classes of GST superfamily, and glutathione S-transferase P1 (GSTP1), glutathione S-transferase theta 1 (GSTT1) and glutathione S-transferase M1 (GSTM1) genes of them are studied extensively for the inter-individual variations of chemotherapeutic effect (Stoehlmacher et al., 2002(Stoehlmacher et al., , 2004)).GSTP1 is reported to be associated with resistance to chemotherapy in vitro studies (Satta et al., 1992;Whelan et al., 1992).GSTP1 was found to be associated with resistance to 5-fluorouracil (5-FU), doxorubicin, mitomycin C and paclitaxel (Su et al., 2003).However, the results of the relationship between GSTP1 and breast cancer with chemotherapy are conflicting (Peters et al., 1993;Schmidt et al., 2003).Therefore, it is needed to explore the association between GSTP1 expression and response to chemotherapy.
In this study, a prospective study to investigate the association of GSTs expression with response to neoadjuvant sequential epirubicin or doxorubicin chemotherapy among breast cancer patients was conducted.

Study population
Primary breast cancer patients ( 219

Response to chemotherapy evaluation
The responses of treatment were evaluated on the basis of standard Response Evaluation Criteria in Solid Tumors (RECIST) criteria.Patients with complete response (CR), partial response (PR), or stable disease remained in the protocol until progressive disease or unacceptable toxicity was documented.Common toxicities were assessed according to the National Cancer Institute Common Toxicity Criteria (NCICTC).

Examination of the GSTP1 polymorphisms
The DNA samples were obtained from stored blood samples using the Qiagen Blood Kit (Qiagen, Chastworth, CA).The GSTP1 I105V polymorphism was examined by the polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) method.0.1 lg of genomic DNA, forward primer 5' -ACC CCA GGG CTC TAT GGG AA-3' and reverse primer 5' -TGA GGG CAC AAG AAG CCC CT-3', were used for PCR amplification.Initial denaturation was carried out at 95°C for 5 min.Cycling conditions were: primer annealing at 55°C for 30 s, polymerization at 72°C for 30 s, and strand separation at 94°C for 30 s. Thirty cycles were carried out.A final polymerization step of 72°C for 5 min was carried out to complete the elongation processes.PCR products, after being digested by BsmAI restriction enzyme (New England Biolabs, Beverly, MA, USA) at 37°C for 2 h, were separated on 2% Nusieve ethidium bromide-stained agarose gels to visualize the bands.

Statistical analysis
The Statistical Package for Social Sciences (SPSS) software version 11.0.1 (SPSS Inc., Chicago, IL, USA) was used for statistical analyses.Association between the various parameters was assessed using Chi-squared test or Fisher's exact test.The association of polymorphisms of GSTP1 with response to chemotherapy in breast cancer patients was calculated by odd ratio (OR) with a corresponding 95% confidence interval (CI).The overall survival curves were plotted using the Kaplan-Meier product limit method, and the statistical differences in survival among subgroups were compared by log-rank test.The relative risk [hazard ratio (HR)] and 95% CI were calculated with the Cox regression model for all significant predictors from cancer diagnosis to the endpoint of the study (event).A primary death from breast cancer was defined as a failure event, and the survival time was defined as the time between diagnosis and death.The cause of death was defined by specialists based on clinical documents and reports by patients' family members.If a patient died from a cause other than breast cancer, her data was censored at the date of death.P-value less than 0.05 was considered statistically significant.

RESULTS
For clinical characteristics, there was no significant difference between patients with GSTP1 There was no significant association between GSTT1 gene polymorphisms and risk of death.

DISCUSSION
The aim of this study was to investigate whether GSTP1 expression was association with the anthracycline-based chemotherapy among breast cancer patients.This study found the GSTP1 expression to be associated with estrogen receptor (ER) status, and was association with previous studies (Miyake et al., 2012).Previous evidences showed that the GSTP1 are involved in response to chemotherapy in various cancers (Ott et al., 2008;Mossallam et al., 2006;Funke et al., 2010;Nagle et al., 2007).However, few studies are conducted in Chinese breast cancer patients.Only several studies conducted in western countries investigation the association of GSTs with chemotherapy response and survival of breast cancer, but the results are conflicting (Oliveira et al., 2010;Mishra et al., 2011;Lourenço et al., 2010).A previous study showed that GSTP1 105 Val may be association with poor survival of breast cancer, and have lower response to chemotherapy treatment for this cancer (Oliveira et al., 2010).However, in another study in Asian population, no significant association was found in GSTP1 and responses to chemotherapy (Mishra et al., 2011), and another study conducted in Japan found a significant responses to chemotherapy patients with GSTP1 Val allele genotype (Ott et al., 2008).This study finds a significant association Zhang et al.The relationship between GSTP1 expression and ER status was found, indicating that the GSTP1 plays a significant role in the suppression of anti-tumor activity of ER status.A previous study indicated the univariate and multivariate analysis of the pathological response to P-FEC showed that only GSTP1 expression was significantly associated with a lower pathological complete response (pCR) rate in ER-negative tumors, but not in ER-positive tumors (Miyake et al., 2012).It has been well established that the pCR rate of ER-positive tumors in response to chemotherapy is lower than that of ER-negative tumors (Kaufmann et al., 2007).In this study, GSTP1 expression was found to be associated with ER status, which indicates that ER had some mechanism between GSTP1 polymorphism and response to chemotherapy.The result is in line with previous ones.

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Conclusively, GSTP1 expression was found to be associated with response to chemotherapy treatment of breast cancer, and ER status had some mechanism between GSTP1 polymorphism and response to chemotherapy.Further prospective studies incorporating larger numbers of patients are needed to validate these associations.
), who were consecutively recruited in this study have been treated with anthracycline-based (epirubicin [E] or doxorubicin [A]) chemotherapy.All the cases were histologically confirmed between March 2006 and March 2007, and the chemotherapy was indicated for breast patients with stage IIA to IIIB.All the patients were treated with NAC.The chemotherapy included anthracycline-based (epirubicin [E] or doxorubicin [A]) chemotherapy.Anthracycline-based chemotherapy consists of cyclophosphamide (C), the anthracycline agent (E or A), and/or 5-fluorouracil (F), (CEF and CAF regimens) combined with radiotherapy.All patients were followed up till March 2011.
IIe/IIe genotype and those with IIe/Val or Val/Val genotype (Table 1).A total of 112 patients carried GSTP1 IIe/IIe genotype and 107 patients carried GSTP1 IIe/Val or Val/Val genotype.

Table 1 .
Association of GSTP1 expression with clinic characteristics of breast cancer.

Table 2 .
Distribution of GSTP1 in responders and non-responders to neoadjuvant chemotherapy for breast cancer.
2Adjusted for age, menopausal status, tumor grade, tumor size, ER, PR and therapeutic regimen.

Table 3 .
Hazard ratios for overall survival in breast cancer patients with chemotherapy.