Tumor Biol. (2014) 35:5561–5567 DOI 10.1007/s13277-014-1732-4
RESEARCH ARTICLE
Genetic polymorphisms in XPG could predict clinical outcome of platinum-based chemotherapy for advanced non-small cell lung cancer Weicai Hu & Jinbing Pan & Pu Zhao & Guangyu Yang & Shujuan Yang
Received: 5 November 2013 / Accepted: 4 February 2014 / Published online: 11 March 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract We conducted a prospective study to investigate the role of four single nucleotide polymorphisms (SNPs) of XPG on the clinical outcome of advanced non-small cell lung cancer (NSCLC) treated with platinum-based doublets chemotherapy. In total, 277 patients with histologically confirmed NSCLC were mainly from December 2007 and December 2008. The genotypes of rs2296147T>C, rs1047768C>T, rs873601G>A, and rs17655G>C were determined by polymerase chain reaction-restriction fragment length polymorphism. By univariate analysis, a shorter survival was associated with older age, sex, and higher disease stage. By multivariate Cox regression analysis, patients carrying rs2296147 TT genotype and T allele were prognostic factors of progression-free survival (PFS) and overall survival (OS). Similarly, patients carrying rs873601 GG genotype and G allele were marginally significantly associated with favorable outcome for PFS and OS. We found that individuals carrying both rs2296147 T allele and rs873601 G allele were associated with better PFS and OS. However, rs1047768C> T and rs17655G>C polymorphisms did not influence the PFS and OS of advanced NSCLC. In summary, our study provided statistical evidence that XPG rs2296147T > C and rs873601G>A polymorphisms may be used as surrogate markers toward individualizing NSCLC treatment strategies. W. Hu : P. Zhao : G. Yang Department of Thoracic Surgery, Henan Provincial People’s Hospital, Zhengzhou, China J. Pan (*) Department of Respiratory Medicine, Henan Provincial People’s Hospital, Zhengzhou, China e-mail: [email protected] S. Yang Department of Health of Social Behavior, West China School of Public Health, Sichuan University, Chengdu, China
Keywords Non-small cell lung cancer . Xeroderma pigmentosum group G . Survival . Progression
Introduction Lung cancer is the most common cause of cancer-related death, and it has been the most common cancer worldwide for several decades [1]. It is estimated that there were 1.8 million new cases in 2012 [1]. Of lung cancer, 70 % of them were non-small cell lung cancer (NSCLC) [2]. Despite a significant improvement in diagnostics, surgery, and chemotherapy, the overall 5-year survival rate is still less than 15 %, and 70 % of them show metastatic disease after treatment [3]. The recommended treatment for NSCLC is surgery, and radiotherapy and chemotherapy are used for unresectable or locally advanced tumors and as palliative therapies for metastatic tumors [4]. Currently, platinum-based chemotherapy, such as cisplatin or carboplatin, is considered as the standard treatment in advanced NSCLC [5, 6]. However, patients with the similar tumor stage and histological type of cancer show different resistance to chemotherapy and different prognosis, which suggests that molecular markers could predict clinical outcomes and treatment sensitivity among NSCLC patients treated with platinum-based doublets chemotherapy. Platinum compounds can induce apoptosis and inhibit the DNA replication in cancer cells by forming the platinumDNA adducts in the DNA [7]. The DNA repair mechanism can allow cancer cell to repair the DNA damages caused by platinum compounds, and it can influence the anticancer effect of these agents [8]. Experimental data indicate that genes in DNA repair system have an important role in cancer susceptibility and increase cancer risk among individuals defective in DNA repair capacity [9]. Nucleotide excision repair (NER) is responsible for repairing platinum-DNA adducts, and 20 enzymes of them are involved in removing and restoring a
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segment of DNA containing a bulky adduct [10, 11]. Single nucleotide polymorphisms (SNPs) in genes involved in NER can influence the efficiency of DNA repair, lead to decreased removal of platinum-DNA adducts, and thus influence the treatment sensitivity of cancer [10, 11]. The XPG (ERCC5) is an indispensable component of NER and is responsible for 11,186 amino acid structure-specific endonuclease activity [10]. The endonuclease XPG is a multifunctional nuclear protein and has an important role in NER of helix-distorting DNA damage and transcription-coupled repair of non-helix distorting DNA lesions [12]. Previous studies reported that SNPs in XPG have an important role in prognosis of various cancers [11, 13–15]. However, several studies reported the association between SNPs in XPG and response to chemotherapy and prognosis in advanced NSCLC, but the results were inconsistent [16–19]. Therefore, we conducted a prospective study to investigate whether the four common SNPs of XPG (rs2296147T>C, rs1047768C>T, rs873601G>A, and rs17655G>C) can help predict survival of advanced NSCLC patients treated with platinum-based chemotherapy.
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Treatment All the patients were treated with platinum-based doublets chemotherapy. The treatment regimens were described in a previous study [20]. The platinum-based doublets chemotherapy was conducted every 3 weeks and repeated for a maximum of six cycles. When patients showed hematological grade three or four drug-related toxicities, the dose of the cytotoxic agents in the next cycle would be reduced by 25 %. The toxicities were followed up to 4 weeks after chemotherapy. Patients were followed up every 4 weeks by telephone or visiting clinics after inclusion into our study until December 2012. The demographic and clinical information of all patients were collected from medical records. Progression-free survival (PFS) and overall survival (OS) were used as endpoints in our study. PFS was defined as the time from enrolling into the study to the time of objective tumor progressive disease. OS was defined as the time from enrolling into the study to the time of death or last clinical follow-up.
SNP selection and genotyping Materials and methods Subjects Two hundred ninety-five eligible patients were diagnosed as unresectable and advanced stage NSCLC (stage III and IV) at Henan Provincial People's Hospital between December 2007 and December 2008. All cases were histopathologically confirmed with no restrictions of age, gender, and histology. Patients who previously received radiotherapy or chemotherapy and had symptomatic brain metastases, spinal cord compression, and uncontrolled massive pleural effusion were excluded from our study. Finally, 277 patients agreed to participate into our study, with a participation rate of 93.90 %. The disease stage of all patients were determined by chest X-ray, chest computed tomography scan, bone scan, and abdominal ultrasonogram. The demographic and clinical information were collected from medical records, including sex, age, smoking status, Eastern Cooperative Oncology Group (ECOG) performance status, disease stage, and tumor histology. The histology and disease stage of NSCLC was based on criteria from World Health Organization. Cigarette smoking was categorized into never, former, and current smokers. Individuals who smoked more than 20 packets of cigarettes per year, or smoked more than one cigarette per day and continued for 6 months were regarded as current smokers, and quit smoking for more than one year were former smokers. All the cases were signed with an informed consent before participating in our study. The protocol was approved by the ethics committee of Henan Provincial People's Hospital.
All patients were asked to provide 5 mL whole blood for genetic testing. The blood samples were stored at −20 °C until use, with 0.5 mg/mL ethylenediaminetetraacetic acid added as an anticoagulant. Extraction of genomic DNA from blood samples was performed using a Qiagen Blood Kit (Qiagen, Chastworth, CA). Four potentially SNPs, rs2296147T>C, rs1047768C>T, rs873601G>A, and rs17655G>C, were selected from NCBI dbSNP database (http://www.ncbi.nlm.nih. gov/). The criteria of selecting SNPs were the following: (1) SNPs at the two ends of XPG, (2) a minor allele frequency (MAF) >5 % in Chinese population, (3) influencing the activity of microRNA binding sites. The probes and primers of rs2296147T > C, rs1047768C > T, rs873601G > A, and rs17655G>C were designed using Sequenom Assay Design 3.1 software (Sequenom). The primers for rs2296147T>C were forward 5’-AGCTGTCACCGCCTCCC-3’ and reverse 5’-CGGCCATTCTCTGGACC-3’, respectively. The primers for rs1047768C>T were forward 5’- CACTTAAAGGAGTC CGA-3’ and reverse 5’-AACTCAATAGAAAATCC-3’, respectively. The primers for rs873601G>A were forward 5’CTGGTATGAGCCCATCTA-3’ and reverse 5’-AGTGACAA GCCTGTAGCC-3’, respectively. The primers for rs17655G> C were 5’-ATCTGGCGGTCACGAGGA-3’ and 5’-CTCCCT ACAATCCTGACTC-3’, respectively. Polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) was developed for detecting the four SNPs. Each PCR reaction mix comprised 25 uL reaction solution with 50 ng genomic DNA, 25 mM MgCl2, 2 mM 4×dNTP, 20 uM primer, and 5 U/μl Taq DNA. The cycling program involved preliminary denaturation at 94 °C for 5 minutes, denatured at 94 °C
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for 45 s, annealed at 62 °C for 60 s and extended at 72 °C for 60 s, and a last extension at 72 °C for 10 minutes. Statistical analysis Continuous variables were expressed as the mean±standard deviation (SD), whereas categorical variables were shown as frequencies and percentages. The survival distributions were estimated using the Kaplan-Meier method. Multivariate Cox proportional hazards model were conducted to evaluate the independent effect of rs2296147T > C, rs1047768C > T, rs873601G > A, and rs17655G>C polymorphisms on OS and PFS with adjusted factors, such as sex, age, smoking, and disease status. Statistical analyses were conducted using the SPSS statistical package, version 11.0 (SPSS Inc., Chicago, IL, USA) for Windows®. All tests were two-tailed with a significance level of 0.05.
Results The main clinical characteristics of enrolled patients are summarized in Table 1. By the end of follow-up, the median follow-up period was 36.4 months, and follow-up period ranged from 2 to 60 months. One hundred ninetysix patients (70.76 %) showed progressive disease and 173 (62.45 %) died until December 2012. The average age was
63.1 years (ranged, 28.7–74.5 years). Among the 277 patients, 184 (66.4 %) were males, 128 (46.2 %) were current and former smokers, 52 (18.8 %) were at ECOG performance stage≥2, 115 (41.5 %) were with squamous cell carcinoma, and 104 (37.5 %) were at disease stage IV. By univariate analysis, a shorter survival was associated with older age, sex, and higher disease stage, with ORs (95 %CI) of 1.72 (1.02–2.89), 1.73 (1.01–2.98), and 11.18 (5.31–25.53), respectively (Table 1). By log-rank test, patients carrying rs2296147 TT genotype and T allele had a significantly longer median PFS and OS than those with CC genotype and C allele (All P valuesC polymorphisms did not influence the PFS and OS of advanced NSCLC. We did not observe a significant interaction between clinical characteristics and rs2296147T >C, rs1047768C >T, rs873601G>A, and rs17655G>C polymorphisms on the clinical outcome of advanced NSCLC. We further conducted the gene-gene interaction between rs2296147T > C and rs873601G > A polymorphisms. We
found that individuals carrying both rs2296147 T allele and rs873601 G allele were associated with better PFS (HR = 0.61, 95 % CI = 0.41–0.90) and OS (HR = 0.43, 95 % CI=0.28–0.65) when compared with those carrying C allele and A allele (Table 3). But no significant interaction was found between rs2296147T > C and rs873601G > A (both P values for interaction > 0.05).
Fig. 2 Kaplan-Meier estimates of OS with rs2296147T > C polymorphism
Fig. 4 Kaplan-Meier estimates of OS with rs873601G > A polymorphism
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Table 2 Univariate analysis of four SNPs in XPG in relation to PFS and OS of advanced NSCLC Gene
Cases
Percent
PFS
OS
Event N=196
Percent
Median (month)
HR (95%CI)a
P value
Event N=173
Percent
Median (month)
HR (95%CI)a
P value
51.62 33.57 14.80 68.41 31.59
116 64 16 296 96
59.18 32.65 8.16 75.51 24.49
16.8 18.7 23.1 17.2 21.5
1.0 (Ref.) 0.85 (0.56–1.29) 0.48 (0.24–0.93) 1.0 (Ref.) 0.52 (0.38–0.70)
– 0.42 0.02 – C CC 143 CT 93 TT 41 C allele 379 T allele 175 rs1047768C>T TT 107 CT 127 CC 44 T allele 341 C allele 214 rs873601G>A AA 98 AG 114 GG 65 A allele 310 G allele 244 rs17655G>C CC CG GG C allele G allele a
74 142 61 290 264
Adjusting sex, age, and disease stage
Discussion Our study found that subjects carrying rs2296147T>C and rs873601G>A polymorphisms were associated with a favorable clinical outcome in advanced NSLCLC treated with platinumbased chemotherapy when compared with wild-type genotypes. Our data indicated that the rs2296147T>C and rs873601G>A polymorphisms were valuable prognostic factors of PFS and OS
of advanced NSCLC, and these gene polymorphisms could be helpful in designing individualized patient treatment. It is generally known that traditional selection of chemotherapy regimen could improve response and survival of cancer patients. Personalized chemotherapy according to molecular biomarkers could further augment response rates and improve survival of NSCLC. Previous published data have indicated that XPG genes are potential predictors for the
Table 3 Interaction between rs2296147T>C and rs873601G>A polymorphisms on PFS and OS of advanced NSCLC Gene
rs2296147T>C C allele T allele C allele T allele a
Cases
rs873601G>A A allele A allele G allele G allele
248 62 131 113
Adjusting sex, age, and disease stage
Percent
44.77 11.19 23.65 20.40
PFS
OS
Event
Percent
HR (95%CI)a
P value
Event
Percent
HR (95%CI)a
P value
184 64 112 51
44.66 15.53 27.18 12.62
1.0 (Ref.) 1.38 (0.92–2.11) 1.15 (0.83–1.60) 0.61 (0.41–0.90)
– 0.10 0.38 0.01
176 41 88 40
50.87 13.01 25.43 10.69
1.0 (Ref.) 0.93 (0.58–1.48) 0.95 (0.67–1.34) 0.43 (0.28–0.65)
– 0.75 0.75 C and rs2094258C>T were associated with clinical outcome of advanced NSCLC [19]. Another study indicated that XPG rs1047768C>T polymorphism was associated with susceptibility to chemotherapy [16]. However, another study did not find that XPG polymorphism may influence overall survival in advanced NSCLC [17]. The possible discrepancy of the results may be induced by the differences in populations, sample size, and study design. Further large sample size studies are greatly needed to confirm the association between XPG polymorphisms and clinical outcome of advanced NSCLC. The current study has three limitations. First, cases were selected from one hospital, which may not be representative of the general population. However, the selection bias may be minimized by further adjustment for potential confounding factors. Second, other genetic polymorphisms may influence the prognosis of NSCLC besides the XPG gene. Third, we did not observe a significant association between rs1047768C>T and rs873601G>A polymorphisms and clinical outcome of advanced NSCLC, and interaction between clinical characteristics and gene polymorphisms. The number of NSCLC cases is relatively small. The relatively small sample size has a limited statistical power to detect a weak effect. These limitations can only be overcome in larger and well-designed studies in the future. In summary, the findings of our study provide statistical evidence that XPG rs2296147T>C and rs873601G>A polymorphisms are associated with clinical outcome of advanced NSCLC patients receiving platinum-based chemotherapy. However, we did not find that rs1047768C>T and rs17655G> C polymorphisms could influence the survival of advanced NSCLC, but the lack of association may be induced by the small sample size. Therefore, further well-designed and large sample
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size studies are greatly needed to confirm the role of XPG polymorphisms and clinical outcome of advanced NSCLC. Acknowledgment The authors would like to thank the Henan Provincial People's Hospital for providing help as well as patients who provided blood samples for our study.
References 1. International Agency for Research on Cancer. Lung cancer estimated incidence, Mortality and Prevalence Worldwide in 2012. 2012. http:// globocan.iarc.fr/factsheet.asp. Access in: 2014-2-1. 2. Ridge CA, McErlean AM, Ginsberg MS. Epidemiology of lung cancer. Semin Interv Radiol. 2013;30(2):93–8. 3. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin. 2010;60(5):277–300. 4. Dy GK, Bogner PN, Tan W, Demmy TL, Farooq A, Chen H, et al. Phase II study of perioperative chemotherapy with cisplatin and pemetrexed in non-small-cell lung cancer. J Thorac Oncol. 2014;9(2):222–30. 5. Bidoli P, Zilembo N, Cortinovis D, Mariani L, Isa L, Aitini E, et al. Randomized phase II three-arm trial with three platinum-based doublets in metastatic non-small-cell lung cancer. An Italian trials in medical oncology study. Ann Oncol. 2007;18(3):461–7. 6. Vilmar A, Sørensen JB. Excision repair cross-complementation group 1 (ERCC1) in platinum-based treatment of non-small cell lung cancer with special emphasis on carboplatin: a review of current literature. Lung Cancer. 2009;64(2):131–9. 7. Johnson SW, Stevenson JP, O’Dwyer PJ. Cisplatin and its analogues. In: DeVita VT, Hellman S, Rosenberg SA, editors. Cancer: principles and practice of oncology. 6th ed. Philadelphia: Lippincott Williams and Wilkins; 2001. p. 376–88. 8. Liu L, Yuan P, Wu C, Zhang X, Wang F, Guo H, et al. Assessment of XPD Lys751Gln and XRCC1 T-77C polymorphisms in advanced non-small-cell lung cancer patients treated with platinum-based chemotherapy. Lung Cancer. 2011;73(1):110–5. 9. Wei Q, Frazier ML, Levin B. DNA repair: a double-edged sword. J Natl Cancer Inst. 2000;92(6):440–1. 10. He C, Duan Z, Li P, Xu Q, Yuan Y. Role of ERCC5 promoter polymorphisms in response to platinum-based chemotherapy in patients with advanced non-small-cell lung cancer. Anticancer Drugs. 2013;24(3):300–5. 11. Fleming ND, Agadjanian H, Nassanian H, Miller CW, Orsulic S, Karlan BY, et al. Xeroderma pigmentosum complementation group C single-nucleotide polymorphisms in the nucleotide excision repair pathway correlate with prolonged progression-free survival in advanced ovarian cancer. Cancer. 2012;118(3):689–97. 12. Melis JP, van Steeg H, Luijten M. Oxidative DNA damage and nucleotide excision repair. Antioxid Redox Signal. 2013;18(18):2409–19. 13. Wen H, Feng CC, Fang ZJ, Xia GW, Jiang HW, Xu G, et al. Study on bladder cancer susceptibility and genetic polymorphisms of XPC, XPG, and CYP in smokers and non-smokers. Actas Urol Esp. 2013;37(5):259–65. 14. Peng Q, Lao X, Li R, Qin X, Li S. Methodological remarks concerning a recent meta-analysis on XPG Asp1104His and XPF Arg415Gln polymorphisms and breast cancer risk. Breast Cancer Res Treat. 2013;139(2):617–8. 15. Yuan H, Li H, Ma H, Niu Y, Wu Y, Zhang S, et al. Genetic polymorphisms in key DNA repair genes and risk of head and neck cancer in a Chinese population. Exp Ther Med. 2012;3(4):719–24. 16. Sun X, Li F, Sun N, Shukui Q, Baoan C, Jifeng F, et al. Polymorphisms in XRCC1 and XPG and response to platinum-
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5567 23. Schrama D, Scherer D, Schneider M, Zapatka M, Bröcker EB, Schadendorf D, et al. ERCC5 p.Asp1104His and ERCC2 p.Lys751Gln polymorphisms are independent prognostic factors for the clinical course of melanoma. J Invest Dermatol. 2011;131(6):1280–90. 24. Ott K, Rachakonda PS, Panzram B, Keller G, Lordick F, Becker K, et al. DNA repair gene and MTHFR gene polymorphisms as prognostic markers in locally advanced adenocarcinoma of the esophagus or stomach treated with cisplatin and 5-fluorouracil-based neoadjuvant chemotherapy. Ann Surg Oncol. 2011;18(9):2688–98. 25. Sakano S, Hinoda Y, Sasaki M, Wada T, Matsumoto H, Eguchi S, et al. Nucleotide excision repair gene polymorphisms may predict acute toxicity in patients treated with chemoradiotherapy for bladder cancer. Pharmacogenomics. 2010;11(10):1377–87. 26. Butkiewicz D, Rusin M, Sikora B, Lach A, Chorąży M. An association between DNA repair gene polymorphisms and survival in patients with resected non-small cell lung cancer. Mol Biol Rep. 2011;38(8):5231–41. 27. Staresincic L, Fagbemi AF, Enzlin JH, Gourdin AM, Wijgers N, Dunand-Sauthier I, et al. Coordination of dual incision and repair synthesis in human nucleotide excision repair. EMBO J. 2009;28(8): 1111–20. 28. Gossage L, Madhusudan S. Current status of excision repair cross complementing-group 1 (ERCC1) in cancer. Cancer Treat Rev. 2007;33:565–77.
RESEARCH ARTICLE
Genetic polymorphisms in XPG could predict clinical outcome of platinum-based chemotherapy for advanced non-small cell lung cancer Weicai Hu & Jinbing Pan & Pu Zhao & Guangyu Yang & Shujuan Yang
Received: 5 November 2013 / Accepted: 4 February 2014 / Published online: 11 March 2014 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract We conducted a prospective study to investigate the role of four single nucleotide polymorphisms (SNPs) of XPG on the clinical outcome of advanced non-small cell lung cancer (NSCLC) treated with platinum-based doublets chemotherapy. In total, 277 patients with histologically confirmed NSCLC were mainly from December 2007 and December 2008. The genotypes of rs2296147T>C, rs1047768C>T, rs873601G>A, and rs17655G>C were determined by polymerase chain reaction-restriction fragment length polymorphism. By univariate analysis, a shorter survival was associated with older age, sex, and higher disease stage. By multivariate Cox regression analysis, patients carrying rs2296147 TT genotype and T allele were prognostic factors of progression-free survival (PFS) and overall survival (OS). Similarly, patients carrying rs873601 GG genotype and G allele were marginally significantly associated with favorable outcome for PFS and OS. We found that individuals carrying both rs2296147 T allele and rs873601 G allele were associated with better PFS and OS. However, rs1047768C> T and rs17655G>C polymorphisms did not influence the PFS and OS of advanced NSCLC. In summary, our study provided statistical evidence that XPG rs2296147T > C and rs873601G>A polymorphisms may be used as surrogate markers toward individualizing NSCLC treatment strategies. W. Hu : P. Zhao : G. Yang Department of Thoracic Surgery, Henan Provincial People’s Hospital, Zhengzhou, China J. Pan (*) Department of Respiratory Medicine, Henan Provincial People’s Hospital, Zhengzhou, China e-mail: [email protected] S. Yang Department of Health of Social Behavior, West China School of Public Health, Sichuan University, Chengdu, China
Keywords Non-small cell lung cancer . Xeroderma pigmentosum group G . Survival . Progression
Introduction Lung cancer is the most common cause of cancer-related death, and it has been the most common cancer worldwide for several decades [1]. It is estimated that there were 1.8 million new cases in 2012 [1]. Of lung cancer, 70 % of them were non-small cell lung cancer (NSCLC) [2]. Despite a significant improvement in diagnostics, surgery, and chemotherapy, the overall 5-year survival rate is still less than 15 %, and 70 % of them show metastatic disease after treatment [3]. The recommended treatment for NSCLC is surgery, and radiotherapy and chemotherapy are used for unresectable or locally advanced tumors and as palliative therapies for metastatic tumors [4]. Currently, platinum-based chemotherapy, such as cisplatin or carboplatin, is considered as the standard treatment in advanced NSCLC [5, 6]. However, patients with the similar tumor stage and histological type of cancer show different resistance to chemotherapy and different prognosis, which suggests that molecular markers could predict clinical outcomes and treatment sensitivity among NSCLC patients treated with platinum-based doublets chemotherapy. Platinum compounds can induce apoptosis and inhibit the DNA replication in cancer cells by forming the platinumDNA adducts in the DNA [7]. The DNA repair mechanism can allow cancer cell to repair the DNA damages caused by platinum compounds, and it can influence the anticancer effect of these agents [8]. Experimental data indicate that genes in DNA repair system have an important role in cancer susceptibility and increase cancer risk among individuals defective in DNA repair capacity [9]. Nucleotide excision repair (NER) is responsible for repairing platinum-DNA adducts, and 20 enzymes of them are involved in removing and restoring a
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segment of DNA containing a bulky adduct [10, 11]. Single nucleotide polymorphisms (SNPs) in genes involved in NER can influence the efficiency of DNA repair, lead to decreased removal of platinum-DNA adducts, and thus influence the treatment sensitivity of cancer [10, 11]. The XPG (ERCC5) is an indispensable component of NER and is responsible for 11,186 amino acid structure-specific endonuclease activity [10]. The endonuclease XPG is a multifunctional nuclear protein and has an important role in NER of helix-distorting DNA damage and transcription-coupled repair of non-helix distorting DNA lesions [12]. Previous studies reported that SNPs in XPG have an important role in prognosis of various cancers [11, 13–15]. However, several studies reported the association between SNPs in XPG and response to chemotherapy and prognosis in advanced NSCLC, but the results were inconsistent [16–19]. Therefore, we conducted a prospective study to investigate whether the four common SNPs of XPG (rs2296147T>C, rs1047768C>T, rs873601G>A, and rs17655G>C) can help predict survival of advanced NSCLC patients treated with platinum-based chemotherapy.
Tumor Biol. (2014) 35:5561–5567
Treatment All the patients were treated with platinum-based doublets chemotherapy. The treatment regimens were described in a previous study [20]. The platinum-based doublets chemotherapy was conducted every 3 weeks and repeated for a maximum of six cycles. When patients showed hematological grade three or four drug-related toxicities, the dose of the cytotoxic agents in the next cycle would be reduced by 25 %. The toxicities were followed up to 4 weeks after chemotherapy. Patients were followed up every 4 weeks by telephone or visiting clinics after inclusion into our study until December 2012. The demographic and clinical information of all patients were collected from medical records. Progression-free survival (PFS) and overall survival (OS) were used as endpoints in our study. PFS was defined as the time from enrolling into the study to the time of objective tumor progressive disease. OS was defined as the time from enrolling into the study to the time of death or last clinical follow-up.
SNP selection and genotyping Materials and methods Subjects Two hundred ninety-five eligible patients were diagnosed as unresectable and advanced stage NSCLC (stage III and IV) at Henan Provincial People's Hospital between December 2007 and December 2008. All cases were histopathologically confirmed with no restrictions of age, gender, and histology. Patients who previously received radiotherapy or chemotherapy and had symptomatic brain metastases, spinal cord compression, and uncontrolled massive pleural effusion were excluded from our study. Finally, 277 patients agreed to participate into our study, with a participation rate of 93.90 %. The disease stage of all patients were determined by chest X-ray, chest computed tomography scan, bone scan, and abdominal ultrasonogram. The demographic and clinical information were collected from medical records, including sex, age, smoking status, Eastern Cooperative Oncology Group (ECOG) performance status, disease stage, and tumor histology. The histology and disease stage of NSCLC was based on criteria from World Health Organization. Cigarette smoking was categorized into never, former, and current smokers. Individuals who smoked more than 20 packets of cigarettes per year, or smoked more than one cigarette per day and continued for 6 months were regarded as current smokers, and quit smoking for more than one year were former smokers. All the cases were signed with an informed consent before participating in our study. The protocol was approved by the ethics committee of Henan Provincial People's Hospital.
All patients were asked to provide 5 mL whole blood for genetic testing. The blood samples were stored at −20 °C until use, with 0.5 mg/mL ethylenediaminetetraacetic acid added as an anticoagulant. Extraction of genomic DNA from blood samples was performed using a Qiagen Blood Kit (Qiagen, Chastworth, CA). Four potentially SNPs, rs2296147T>C, rs1047768C>T, rs873601G>A, and rs17655G>C, were selected from NCBI dbSNP database (http://www.ncbi.nlm.nih. gov/). The criteria of selecting SNPs were the following: (1) SNPs at the two ends of XPG, (2) a minor allele frequency (MAF) >5 % in Chinese population, (3) influencing the activity of microRNA binding sites. The probes and primers of rs2296147T > C, rs1047768C > T, rs873601G > A, and rs17655G>C were designed using Sequenom Assay Design 3.1 software (Sequenom). The primers for rs2296147T>C were forward 5’-AGCTGTCACCGCCTCCC-3’ and reverse 5’-CGGCCATTCTCTGGACC-3’, respectively. The primers for rs1047768C>T were forward 5’- CACTTAAAGGAGTC CGA-3’ and reverse 5’-AACTCAATAGAAAATCC-3’, respectively. The primers for rs873601G>A were forward 5’CTGGTATGAGCCCATCTA-3’ and reverse 5’-AGTGACAA GCCTGTAGCC-3’, respectively. The primers for rs17655G> C were 5’-ATCTGGCGGTCACGAGGA-3’ and 5’-CTCCCT ACAATCCTGACTC-3’, respectively. Polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) was developed for detecting the four SNPs. Each PCR reaction mix comprised 25 uL reaction solution with 50 ng genomic DNA, 25 mM MgCl2, 2 mM 4×dNTP, 20 uM primer, and 5 U/μl Taq DNA. The cycling program involved preliminary denaturation at 94 °C for 5 minutes, denatured at 94 °C
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for 45 s, annealed at 62 °C for 60 s and extended at 72 °C for 60 s, and a last extension at 72 °C for 10 minutes. Statistical analysis Continuous variables were expressed as the mean±standard deviation (SD), whereas categorical variables were shown as frequencies and percentages. The survival distributions were estimated using the Kaplan-Meier method. Multivariate Cox proportional hazards model were conducted to evaluate the independent effect of rs2296147T > C, rs1047768C > T, rs873601G > A, and rs17655G>C polymorphisms on OS and PFS with adjusted factors, such as sex, age, smoking, and disease status. Statistical analyses were conducted using the SPSS statistical package, version 11.0 (SPSS Inc., Chicago, IL, USA) for Windows®. All tests were two-tailed with a significance level of 0.05.
Results The main clinical characteristics of enrolled patients are summarized in Table 1. By the end of follow-up, the median follow-up period was 36.4 months, and follow-up period ranged from 2 to 60 months. One hundred ninetysix patients (70.76 %) showed progressive disease and 173 (62.45 %) died until December 2012. The average age was
63.1 years (ranged, 28.7–74.5 years). Among the 277 patients, 184 (66.4 %) were males, 128 (46.2 %) were current and former smokers, 52 (18.8 %) were at ECOG performance stage≥2, 115 (41.5 %) were with squamous cell carcinoma, and 104 (37.5 %) were at disease stage IV. By univariate analysis, a shorter survival was associated with older age, sex, and higher disease stage, with ORs (95 %CI) of 1.72 (1.02–2.89), 1.73 (1.01–2.98), and 11.18 (5.31–25.53), respectively (Table 1). By log-rank test, patients carrying rs2296147 TT genotype and T allele had a significantly longer median PFS and OS than those with CC genotype and C allele (All P valuesC polymorphisms did not influence the PFS and OS of advanced NSCLC. We did not observe a significant interaction between clinical characteristics and rs2296147T >C, rs1047768C >T, rs873601G>A, and rs17655G>C polymorphisms on the clinical outcome of advanced NSCLC. We further conducted the gene-gene interaction between rs2296147T > C and rs873601G > A polymorphisms. We
found that individuals carrying both rs2296147 T allele and rs873601 G allele were associated with better PFS (HR = 0.61, 95 % CI = 0.41–0.90) and OS (HR = 0.43, 95 % CI=0.28–0.65) when compared with those carrying C allele and A allele (Table 3). But no significant interaction was found between rs2296147T > C and rs873601G > A (both P values for interaction > 0.05).
Fig. 2 Kaplan-Meier estimates of OS with rs2296147T > C polymorphism
Fig. 4 Kaplan-Meier estimates of OS with rs873601G > A polymorphism
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Table 2 Univariate analysis of four SNPs in XPG in relation to PFS and OS of advanced NSCLC Gene
Cases
Percent
PFS
OS
Event N=196
Percent
Median (month)
HR (95%CI)a
P value
Event N=173
Percent
Median (month)
HR (95%CI)a
P value
51.62 33.57 14.80 68.41 31.59
116 64 16 296 96
59.18 32.65 8.16 75.51 24.49
16.8 18.7 23.1 17.2 21.5
1.0 (Ref.) 0.85 (0.56–1.29) 0.48 (0.24–0.93) 1.0 (Ref.) 0.52 (0.38–0.70)
– 0.42 0.02 – C CC 143 CT 93 TT 41 C allele 379 T allele 175 rs1047768C>T TT 107 CT 127 CC 44 T allele 341 C allele 214 rs873601G>A AA 98 AG 114 GG 65 A allele 310 G allele 244 rs17655G>C CC CG GG C allele G allele a
74 142 61 290 264
Adjusting sex, age, and disease stage
Discussion Our study found that subjects carrying rs2296147T>C and rs873601G>A polymorphisms were associated with a favorable clinical outcome in advanced NSLCLC treated with platinumbased chemotherapy when compared with wild-type genotypes. Our data indicated that the rs2296147T>C and rs873601G>A polymorphisms were valuable prognostic factors of PFS and OS
of advanced NSCLC, and these gene polymorphisms could be helpful in designing individualized patient treatment. It is generally known that traditional selection of chemotherapy regimen could improve response and survival of cancer patients. Personalized chemotherapy according to molecular biomarkers could further augment response rates and improve survival of NSCLC. Previous published data have indicated that XPG genes are potential predictors for the
Table 3 Interaction between rs2296147T>C and rs873601G>A polymorphisms on PFS and OS of advanced NSCLC Gene
rs2296147T>C C allele T allele C allele T allele a
Cases
rs873601G>A A allele A allele G allele G allele
248 62 131 113
Adjusting sex, age, and disease stage
Percent
44.77 11.19 23.65 20.40
PFS
OS
Event
Percent
HR (95%CI)a
P value
Event
Percent
HR (95%CI)a
P value
184 64 112 51
44.66 15.53 27.18 12.62
1.0 (Ref.) 1.38 (0.92–2.11) 1.15 (0.83–1.60) 0.61 (0.41–0.90)
– 0.10 0.38 0.01
176 41 88 40
50.87 13.01 25.43 10.69
1.0 (Ref.) 0.93 (0.58–1.48) 0.95 (0.67–1.34) 0.43 (0.28–0.65)
– 0.75 0.75 C and rs2094258C>T were associated with clinical outcome of advanced NSCLC [19]. Another study indicated that XPG rs1047768C>T polymorphism was associated with susceptibility to chemotherapy [16]. However, another study did not find that XPG polymorphism may influence overall survival in advanced NSCLC [17]. The possible discrepancy of the results may be induced by the differences in populations, sample size, and study design. Further large sample size studies are greatly needed to confirm the association between XPG polymorphisms and clinical outcome of advanced NSCLC. The current study has three limitations. First, cases were selected from one hospital, which may not be representative of the general population. However, the selection bias may be minimized by further adjustment for potential confounding factors. Second, other genetic polymorphisms may influence the prognosis of NSCLC besides the XPG gene. Third, we did not observe a significant association between rs1047768C>T and rs873601G>A polymorphisms and clinical outcome of advanced NSCLC, and interaction between clinical characteristics and gene polymorphisms. The number of NSCLC cases is relatively small. The relatively small sample size has a limited statistical power to detect a weak effect. These limitations can only be overcome in larger and well-designed studies in the future. In summary, the findings of our study provide statistical evidence that XPG rs2296147T>C and rs873601G>A polymorphisms are associated with clinical outcome of advanced NSCLC patients receiving platinum-based chemotherapy. However, we did not find that rs1047768C>T and rs17655G> C polymorphisms could influence the survival of advanced NSCLC, but the lack of association may be induced by the small sample size. Therefore, further well-designed and large sample
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size studies are greatly needed to confirm the role of XPG polymorphisms and clinical outcome of advanced NSCLC. Acknowledgment The authors would like to thank the Henan Provincial People's Hospital for providing help as well as patients who provided blood samples for our study.
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