Glutathione S-transferase P1 and DNA Polymorphisms with the Response to Chemotherapy and the Prognosis of Bone Tumor

Osteosarcoma derives from primitive bone-forming mesenchymal cells and is the most common primary bone malignancy in children and adolescents. The risk of being diagnosed with cancer increases as an individual ages, and 77% of all cancers are diagnosed in persons aged 55 years and above. As a lifetime risk, the probability that an individual, over the course of a lifetime, will develop a cancer is slightly less than one in two for men and a little more than one in three for women (US Cancer Statistics Working Group, 2007; Ries et al., 2009). The etiology of OS still remains unknown, and the progression might be influenced by the genetic factors (Fuchs et al., 2001). Standard treatment of osteosarcoma is based on a combination of different drugs: neoadjuvant therapy with methotrexate, cisplatin, and adriamycin followed by surgery and post-operative chemotherapy (methotrexate, cisplatin, adriamycin, cyclophospharmide, and vinciristine). Nevertheless, multi-drug resistance and poor clinical outcome are the main problems of 50% osteosarcoma patients. Clinical response to chemotherapeutics is a complex trait that is influenced by genetic and environmental factors. Anticancer therapies have a narrow therapeutic range so that a higher concentration in patient’s body causes toxicity and a lower concentration reduces the


Introduction
Osteosarcoma derives from primitive bone-forming mesenchymal cells and is the most common primary bone malignancy in children and adolescents. The risk of being diagnosed with cancer increases as an individual ages, and 77% of all cancers are diagnosed in persons aged 55 years and above. As a lifetime risk, the probability that an individual, over the course of a lifetime, will develop a cancer is slightly less than one in two for men and a little more than one in three for women (US Cancer Statistics Working Group, 2007;Ries et al., 2009). The etiology of OS still remains unknown, and the progression might be influenced by the genetic factors (Fuchs et al., 2001). Standard treatment of osteosarcoma is based on a combination of different drugs: neoadjuvant therapy with methotrexate, cisplatin, and adriamycin followed by surgery and post-operative chemotherapy (methotrexate, cisplatin, adriamycin, cyclophospharmide, and vinciristine). Nevertheless, multi-drug resistance and poor clinical outcome are the main problems of 50% osteosarcoma patients.
Clinical response to chemotherapeutics is a complex trait that is influenced by genetic and environmental factors. Anticancer therapies have a narrow therapeutic range so that a higher concentration in patient's body causes toxicity and a lower concentration reduces the

Glutathione S-transferase P1 and DNA Polymorphisms with the Response to Chemotherapy and the Prognosis of Bone Tumor
Li-Min Yang 1 , Xiu-Hua Li 1 *, Cui-Fen Bao 2 efficacy of the drug. Interindividual differences in pharmacokinetics and pharmacodynamics determine the global response and toxicity profile of each drug. In this process, the genes involved are the ones that control drug absorption, distribution, metabolism, and excretion. Our previous study indicated ERCC1 and ERCC2 might have a role in the prognosis of bone tumor (Hao et al., 2012). Glutathione S-transferases (GSTs) are a family of cytosolic enzymes involved in the detoxification of various exogenous as well as endogenous reactive species (Ketterer, 1998;Hengstler et al., 1998). GSTs function as dimers by catalyzing the conjugation of mutagenic electrophilic substrates to glutathione. The coding region polymorphisms within GSTP1 have been suggested to confer different catalytic activities (Zimniak et al., 2002). The effects of these polymorphisms on drug metabolism, including chemotherapeutic agents, make these genes candidates for investigation of toxicity and resistance mechanisms (Wang et al., 2002).
Pharmacogenetic studies have shown that germline polymorphisms in genes related to drug metabolism and transport can have a major effect on the pharmacokinetics and pharmacodynamics of these drugs (Zhou et al., 2008). We previously performed a study of the nucleotide excision DNA repair pathway in relation to response to cisplatin and observed an association between osteosarcoma outcome and a polymorphism in ERCC1 and ERCC2 (Hao et al., 2012). In this study we studies a comprehensive set of three SNPs and identified the prognostic genetic factors for the chemotherapeutic agents, and their association with drug response.

Materials and Methods
187 patients diagnosed with osteosarcoma were collected at department of traumatic orthopedics of Zhengzhou hospital, 91 Central Hospital of PLA, Children Hospital of Zhengzhou and the Second Affiliated Hospital of Inner Mongolia Medical University between January 2005 and January 2007. All the included cases in our study were histologically confirmed. All interviews and blood samples collection were conducted after obtaining signed informed consent from participants. Patients were treated with neoadjuvant chemotherapy based on doxorubicin, methotrexate, cisplatin and ifosfamide before and after surgery.
Patients were subsequently grouped as responders (complete+partial response) or nonresponders (stable+progressive disease). Cases with secondary or recurrent tumors were excluded. All patients were followed up till January 2012.

DNA extraction and quantification
The DNA samples were obtained from peripheral blood lymphocytes by using the Qiagen Blood Kit (Qiagen, Chastworth, CA). Genotyping for GSTP1, ERCC1 and ERCC2 polymorphisms by PCR-RFLP assay was done following a modified method of Ateş et al (Ateş et al., 2005). The primer sequences of GSTP1, ERCC1 and ERCC2 polymorphisms were showed in Table 1. Polymerase chain reaction was started at 94°C for 4 min, followed by 35 cycles of 94°C for 30 s, 61°C for 30 s, and 72°C for 30 s, and a final elongation step at 72°C for 7 min. We also performed the genotyping of internal positive control samples, use of no template controls, and use of replicates for 10% samples for quality control. These results of the quality control analysis confirmed 100% concordance.

Statistical analysis
The SPSS software package version 16.0 (SPSS Inc. Chicago, USA) was used for statistical analysis. Association between the various parameters was assessed by using Chi-squared test or Fisher's exact test. The association of polymorphisms of genotypes with response to chemotherapy was calculated by odds ratios (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 correlations between different genotypes of GSTP1, ERCC1 and ERCC2 and response were analyzed by using multiple logistic analyses. The independent prognostic values of different polymorphism including GSTP1, ERCC1 and ERCC2 their association with survival of bone cancer were analyzed by Cox Hazard regression model. Cox hazard ratio and its 95% confidence interval (CI) were analyzed accordingly. P-value less than 0.05 was considered statistically significant.

Results
The clinical features of 187 osteosarcoma patients were showed in Table 2. The median age at diagnosis is 17.7±9.6 years (range: 7-39 years old). Most of the patients were less than 30 years, and more than 50% of the patients were at the range of 10-30 years old. The diagnosis was established with histological examination in all cases. Among 187 patients, most of them were males (56.68%). 55.08% of the osteosarcoma were osteoblastic, and 18.18% of them were chondroblastic. The remainders were regarded as other type. When the cases included in our study, 20.32% of the patients were metastasis, whereas 27.81% of them had developed metastasis since January 2012.
Among 187 patients, 97 patients showed response to neoadjuvant chemotherapy. Among 97 responders, 17.53% took GSTP1 Val/Val genotype, 12.37% took ERCC1 T/T genotype and 9.28% took ERCC2 A/A genotype (Table  3). GSTP1 Val/Val genotype had significantly higher rates of response to chemotherapy when compared to GSTP1 Ile/ Ile genotype, and the adjusted OR (95% CI)     table 4. Polymorphisms in GSTP1 Val/Val and ERCC1 T/T had a higher survival rate than nonnull genotype, whereas no significant association was found between these two genotypes and prognosis of osteosarcoma with neoadjuvant chemotherapy(For GSTP1 Val/Val, HR=0.53, 95%CI: 0.24-1.16; for ERCC1 T/T, HR=0.74, 95%CI: 0.28-1.94). Individuals with ERCC2 A/A genotype were likely to have a lower risk of death from oseosarcoma, and they were tended to live longer than ERCC2 G/G genotype (Figure 1). A significantly adjusted Hazard ratio was found between the ERCC2 A/A and prognosis of osteosarcoma (adjusted HR=0.32, 95% CI=0.13-0.95).

Discussion
In the present study, we provide evidence that glutathione S-transferases and DNA repaired gene are involved in response to adjuvant chemotherapy in Chinese osteosarcoma patients. Our results showed a significant relationship with response to chemotherapy among individuals with GSTP1 Val/Val and ERCC2 A/A genotypes. Results of various subsequent studies have shown that consistent of nature of this relationship (Riddick et al., 2005). A similar association is found in of 2.19(1.15-6.21). ERCC2 A/A genotype also presented a higher rate of response to chemotherapy (OR=2.88, 95% CI=1.14-13.25). However, we did not find a significant risk of response to neoadjuvant chemotherapy with other genotypes among osteosarcoma patients.
The average survival time of 187 patients were 38.4 months. The association between GSTP1, ERCC1 and ERCC2 gene polymorphisms with prognosis of previous studies (Pasello et al., 2008;Pasello et al., 2011;Windsor et al., 2012;Zhang et al., 2012). Zhang et al., in 2012, studies 159 patients with osteosarcoma treated with neoadjuvant chemotherapy and did not find any association of increased risk of death from osteosarcoma among patients with GSTP1 Val/Val genotype . However, another two studies conducted in Italy reported GSTP1 Val/Val was associated with the outcome of osteosarcoma, and GSTP1 could be considered a promising new therapeutic possibility for osteosarcoma patients (Pasello et al., 2008;Pasello et al., 2011). The inconsistencey of these results may be explained by differences in different ethnicities, source of control, sample size and etc. Further confirmation of these results is needed in future studies.
Numbers of studies have suggested an important role for drug-metabolizing enzymes in determining interindividual variations in therapeutic response to chemotherapy among cancer patients. GSTs make up a family of multifunctional enzymes that detoxify a variety of eletrophilic compounds. The explanation of polymorphisms in GSTP1 gene on the efficacy of detoxifying cytotoxins generated by chemotherapeutics might be impairment of the GSTP1 capacity caused by the AgG substitution, patients with the Val variant allele may be less capable of detoxifying oxaliplatin compared to patients with wide-type allele. Our study indicated GSTP1 Val/Val has forable overall survival, which is agreement with previous reports in various cancers, including breast, colorectal (Stoehlmacher et al., 2002;Kweekel et al., 2008). ERCC1 and ERCC2 are potentially relevant to cancer because of their involvement in the process of nucleotide expcision repair (NER) (Goode et al., 2002), and the NER acts a role in repairmen of DNA damaged by environment and UV damage, and the polymorphisms in the two DNA repared genes may change the function of NER in repairing DNA damage by chemotherapy. Our study indicated ERCC2 A/A genotype also presented a higher rate of response to chemotherapy, and ERCC2 A/A was significantly associated with higher risk of death from osteosarcoma. Previous studies showed ERCC2 was associated with response to chemotherapy among osteosarcoma (Caronia et al., 2009), and survival of this cancer (Hao et al., 2012). Our results here are in line with these previous studies.
We found polymorphisms in GSTP1 and ERCC2 could modify the chemotherapy of patients with osteosarcoma, and they also have an important role in their prognosis. However, there was no significant association of ERCC1 C118T with osteosarcoma. We conclude that the test of GSTP1 and ERCC2 Lys751Gln polymorphisms might be a candidate pharmacogenomic factors to be explored in the future to identify the osteosarcoma patients who might benefit from chemotherapy.