Prognostic importance of DNA repair gene ... - Springer Link

J Cancer Res Clin Oncol (2008) 134:645–652 DOI 10.1007/s00432-007-0328-4

ORIGINAL PAPER

Prognostic importance of DNA repair gene polymorphisms of XRCC1 Arg399Gln and XPD Lys751Gln in lung cancer patients from India Leelakumari Sreeja · Volga S. Syamala · Vani Syamala · Sreedharan Hariharan · Praveenkumar B. Raveendran · R. V. Vijayalekshmi · Jayaprakash Madhavan · Ravindran Ankathil

Received: 5 April 2007 / Accepted: 28 September 2007 / Published online: 19 October 2007 © Springer-Verlag 2007

Abstract Purpose Inter individual variation in lung cancer susceptibility may be modulated in part through genetic polymorphisms in the DNA repair genes, especially the genes involved in the Base Excision Repair (BER) and nucleotide excision repair (NER) pathway. Two of the genetic polymorphisms, XRCC1Arg399Gln and XPD Lys751Gln have been extensively studied in the association with lung cancer risk, although published studies have been inconclusive. Methods In order to verify the role of the common variant alleles in the XPD gene, we have genotyped 211 lung cancer patients and 211 healthy controls using PCR-RFLP assays in a hospital based, case-control study in an Indian population. Logistic regression models were Wt to examine the relationship between the log odds of lung cancer and each covariate. Overall Survival in relation to various genotypes and clinicopathological factors were analyzed using Kaplan Meier estimates and hazard ratios were calculated using Cox Regression analysis. Results The carriers of XRCC1 399 AA genotypes were at higher risk of lung cancer (OR = 2.1, 95% CI:1.224–3.669, P = 0.007) than carriers of GG genotype. Subjects carrying

751 AC genotype were at an increased risk of carcinoma of the lung (OR = 1.8; 95% CI:1.233–2.807, P = 0.003) than subjects with AA genotypes. Compared to the XRCC1 399 GG/ XPD 751 AA reference genotype, the combined variants, XRCC1 399 GG/ XPD 751 AC+CC (OR = 1.9, 95% CI: 1.037–3.481), P = 0.03), XRCC1 399 GA+AA/ XPD 751 AA (OR = 1.7, 95% CI: 1.020–2.833, P = 0.04), XRCC1 399 GA+AA/XPD 751 AC+CC (OR = 2.7, 95% CI: 1.582–4.864, P = 0.01), had signiWcantly higher odds ratios. Increasing numbers of either XPD or XRCC1 variant alleles were associated with shorter overall survival, the risk being signiWcant for the XRCC1 gene polymorphism (P = 0.01 by log-rank test). The hazard of dying was signiWcant for the XRCC1 399 AA genotype (HR = 3.04, 95%CI: 1.393–6.670, P = 0.005). Higher tumour stage also came out as signiWcant predictors of patient death. Conclusions These Wndings suggest that genetic polymorphisms in the DNA repair genes may modulate overall lung cancer susceptibility and that pathological stage and XRCC1 Arg399Gln independently predicted overall survival among Indian lung cancer patients. Keywords DNA repair · XRCC1 · XPD · Susceptibility · Combination genotypes · Stage · Survival

L. Sreeja · V. S. Syamala · V. Syamala · S. Hariharan · P. B. Raveendran · R. V. Vijayalekshmi · J. Madhavan · R. Ankathil (&) Division of Cancer Research, Regional Cancer Centre, Trivandrum, Kerala 695011, India e-mail: [email protected] L. Sreeja e-mail: [email protected] J. Madhavan Division of Radiation Oncology, Regional Cancer Centre, Trivandrum, Kerala 695011, India

Introduction DNA repair genes play a major role in maintaining genomic stability through diVerent pathways that are mediated by base excision and nucleotide excision genes. Polymorphisms in DNA repair genes may be associated with diVerences in the repair eYciency of DNA damage and may inXuence an individual’s risk of lung cancer. It may result in subtle structural alteration of the repair enzymes and

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modulation of cancer susceptibility (Benhamou and Sarasin 2000). The genes belonging to Base excision repair (BER) and nucleotide excision repair (NER) pathway, such as Xray Repair Cross Complementing Group 1 (XRCC1) and XP Complementation group D (Xeroderma Pigmentosum group D, XPD) have been extensively studied in the association with lung cancer. The XRCC1 gene product plays an important role in the BER pathway by acting as a scaVold for other DNA repair proteins, such as DNA polymerase (Kubota et al. 1996) and DNA ligase III (Caldecott 2003). Genetic polymorphisms of the XRCC1 gene have been identiWed at codon 194 (C > T substitution at position 26304, exon 6, Arg to Trp), codon 280 (G > A substitution at position 27466, exon 9, Arg to His) and 399 (G > A substitution at position 28152, exon 10, Arg to Gln). The NER pathway repairs a wide variety of DNA damage, including cross-links, oxidative damage and bulky adducts (such as polycyclic aromatic hydrocarbon-DNA adducts). The XPD (XP Complementation group D) gene encodes a helicase, a major DNA repair protein, which is involved in transcription-coupled NER and in the removal of a variety of structurally unrelated DNA lesions (Lehmann 2001) including those induced by tobacco carcinogens (Tang et al. 2002; Leadon and Cooper 1993). Several non-synonymous single nucleotide polymorphisms (SNPs) have been described in the XPD gene, including those at codon 312 (G > A substitution at position 23591, exon 10, Asp > Asn) and codon 751 (A > C substitution at position 35931, exon 23, Lys > Gln). Within this pathway, plausible candidate susceptibility gene is the common XPD codon 751 polymorphism (Shen et al. 1998). The normal functioning XPD protein plays an essential role in NER and participates in the unwinding of DNA at the site of a deleterious DNA lesion (Sung et al. 1993; Hoeijmakers et al. 1996). The XRCC1 Arg399Gln polymorphism is located in the area coding for a PARP binding site. PARP is a zinc-Wnger containing enzyme that detects DNA strand breaks (Shall and de Murcia 2000). Carriers of the XRCC1 399 Gln variant allele have been shown to have higher levels of DNA adducts (Lunn et al. 1999) and to be at greater risk for ionizing radiation sensitivity (Hu et al. 2001) and tobaccorelated DNA damage (Duell et al. 2000; Abdel-Rahman and El Zein 2000; Lei et al. 2002). Moreover, several studies showed that the XPD codon 751 polymorphism is associated with lung cancer (Butkiewicz et al. 2001; Spitz et al. 2001). This polymorphism appears to be connected with smoking status and may increase cancer risk among nonsmokers (Zhou et al. 2002a). Although the functional signiWcance of these polymorphisms has not yet been elucidated, it is possible that they may be responsible, in part, for the inter individual DNA damage repair variations in the general population and for a low DNA repair capacity phenotype characteristic of cancer patients (Lunn et al.

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J Cancer Res Clin Oncol (2008) 134:645–652

2000).To test this hypothesis, we performed a hospital based case-control study using a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay to genotype two DNA repair genes XRCC1 Arg399Gln and XPD Lys751Gln polymorphisms in a casecontrol study in an Indian population, in relation to lung cancer susceptibility and overall survival.

DNA extraction and genotyping Blood samples of all study subjects were collected in 5 ml ACD tubes and stored at ¡80°C until use. Genomic DNA was isolated using Phenol–Chloroform extraction method. Genotypic analysis of the XRCC1 399 G > A polymorphism was determined by the PCR-RFLP method, as described in detail earlier (Lunn et al. 2000). BrieXy, PCR primers for the XRCC1 codon 399 (forward 5⬘-TTGTGCT TTCTCTGTGTCCA-3⬘ and reverse 5⬘-TCCTCCAGCCTT TTCTGATA-3⬘) were used to generate a 615-bp product containing the polymorphic sites. The PCR reactions were carried out in a 20-l volume of 20 pmol of each primer, 0.2 mM each dNTP and 1£ buVer and 1 U of Taq polymerase, with a denaturation of 94°C for 5 min, followed by 30 cycles of 30 s at 61°C and 45 s at 72°C and Wnally 7 min at 72°C. Following ampliWcation, PCR products were digested using 10 U of restriction enzyme Msp I (New England BioLabs, Beverly, MA, USA) for 16 h at 37°C, and electrophoresed on a 3% agarose gel. The allele types were determined as follows: two fragments of 221 and 374 bp for the GG genotype, three fragments of 615, 374 and 221 bp for the GA genotype and a single 615 bp fragment for the AA genotype. The XPD 751 A > C polymorphism was also determined by PCR-RFLP method using speciWc primers (forward, 5⬘-GCCCGCTCTGGATTAT ACG-3⬘ and reverse, 5⬘-CTATCATCTCCTGGCCCCC-3⬘ as described (Tomescu et al. 2001). PCR reaction was performed in a 20-l volume containing 1.5 mM MgCl2, 0.2 mM dNTP, 3% dimethyl sulfoxide, 0.2 M primers, 100 ng template DNA and 1.5 U Taq polymerase in 1£ PCR buVer, 50 mM KCl and 0.1% Triton X-100. Initial denaturation at 94°C for 3 min was followed by 38 cycles of 45 s at 94°C, 45 s at 60°C and 60 s at 72°C, and then a Wnal extension step of 7 min at 72°C. Following PCR, 20 l of PCR product was subjected to restriction digestion using 10 U of Pst I restriction enzyme (New England BioLabs, USA). The digested products were resolved on 3.0% agarose gel and stained with 0.5 g/ml ethidium bromide. The restriction products of the XPD codon 751 AA, AC and CC genotypes had band sizes of 234/110, 234/171/110/63 and 110/63 bp, respectively. For quality control, a random 5% of the samples were repeated and all the inconclusive samples were reanalysed.


647

Statistical methods The odds ratio (OR) and their 95% conWdence intervals (CI) for the genotypes were calculated by unconditional logistic regression analysis, after adjusting for possible confounding factors such as age, gender and smoking status, to estimate the strength of the association between the genotypes and lung cancer risk. The genotypes were categorized into three groups (major allele homozygous, heterozygous and homozygous variant keeping the major allele as the reference group). Probability value at the 0.05). Regarding the distribution of XRCC1 Arg399Gln genotypes in the study population, in cases 37% were GG, 40.8% GA and 22.3% GG when compared to 48.3, 37.9 and 13.7% in controls. The distribution of genotypes for XPD Lys751Gln polymorphism were 51.7% 751 AA, 42.2% AC and 6.2% CC respectively. SigniWcantly higher odds ratios were obtained for XRCC1 399 AA genotype (OR = 2.1, 95% CI = 1.224– 3.669, P = 0.007) as well as XPD 751AC genotype with an OR of 1.86 (95% CI = 1.233–2.807, P = 0.003) compared to their normal counterparts (Table 2). We also looked for the combined eVect of these genes in lung cancer susceptibility. Compared to the XRCC1 399 GG/XPD 751 AA genotype which being the reference genotype, the other combined variants namely, XRCC1 399 GG/XPD 751 AC + CC (OR = 1.9, 95% CI = 1.037–3.481, P = 0.03), XRCC1 399 GA + AA/XPD 751 AA (OR = 1.7, 95% CI = 1.020–2.833, P = 0.04), XRCC1 399 GA + AA/XPD Table 2 Association between XRCC1 Arg399Gln and XPD Lys751Gln genotypes either singly or in combinations to development of lung cancer

751 AC + CC (OR = 2.7, 95% CI = 1.582–4.864, P = 0.01), had signiWcantly higher odds ratios (Table 2). Clinical characteristics of the patients were compared across the XRCC1 399 and XPD 751 genotypes. There were no signiWcant diVerences in the genotype distributions by clinicopathological features of lung cancer like stage, histology or metastasis (Data not shown). The associations of XRCC1 and XPD genotypes either singly or in combinations as well as clinicopathological features in lung cancer patients with survival were also tested. Data of 170 patients were available for the survival analysis, of which there were 66 deaths (Table 3). The median survival of the patients was 10 months. For patients possessing XRCC1 GG genotype, the median survival was 31months (95% CI = 17–45). Also, those with XRCC1 399 GA genotype had a median survival of 21 months (95% CI = 17.16– 24.84) and XRCC1 399 AA genotype had 14 months (95% CI = 7.98–16.02). Hence the patients possessing XRCC1 399 AA genotype had poorer prognosis (P = 0.01 by log rank test) (Fig. 1). Patients with XPD 751 AA genotype had

Cases/control

OR (95% CI)a

XRCC1 399 GG

78/102

Reference

XRCC1 399 GA

86/80

1.4 (0.920–2.148)

0.11

XRCC1 399 AA

47/29

2.1 (1.224–3.669)

0.007

XPD 751 AA

109/139

Reference

XPD 751 AC

89/61

1.8 (1.233–2.807)

0.003

XPD 751 CC

13/11

1.5 (0.650–3.495)

0.33

40/68

Reference

P-value

Genotype

Genotype combinations XRCC1 399 GG/XPD 751 AA

a

Odds ratio adjusted for age, gender and smoking

XRCC1 399 GG/XPD 751 AC + CC

38/34

1.9 (1.037–3.481)

0.03

XRCC1 399 GA + AA/XPD 751 AA

71/71

1.7 (1.020–2.833)

0.04

XRCC1 399 GA + AA/XPD 751 AC + CC

62/38

2.7 (1.582–4.864)

0.01

Table 3 XRCC1 399 and XPD 751 genotypes and clinical features in relation to overall survival in patients with lung cancer polymorphisms and clinicopathological factors with respect to overall survival Parameter

Events/censored

Median survival

XRCC1 399 GG

16/47

31

XRCC1 399 GA

31/40

21

Log rank P-value

Hazard ratio (95% CI) Reference

0.01

1.87 (0.941–3.755)

0.07 0.005

XRCC1 399 AA

19/17

12

3.04 (1.393–6.670)

XPD 751 AA

29/60

23

Reference

XPD751 AC

30/40

20

XPD 751 CC

7/4

14

Stage I and II

24/49

31

Stage III and IV

37/37

14

2.1 (1.089–4.403)

No metastasis

45/86

31

1

Metastasis

21/18

15

123

P-value

0.19 0.002

0.02

1.34 (0.767–2.368)

0.29

2.45 (0.957–6.302)

0.06

Reference

1.08 (0.556–2.127)

0.02 0.80


649 1.2

1.2 Log Rank P Value =0.01

1.0

1.0

Cumulative survival

Cumulative Survival

Log Rank P Value =0.002 .8

.8 .6 XRCC1 Arg399Arg

.4 XRCC1 Gln399Gln

.2

.4

Stage I & II

.2

Stage III & IV

0.0

XRCC1 Arg399Gln

0.0

.6

-.2

-.2

0

0

20

40

60

80

100

Survival in months

Fig. 1 Kaplan Meier Survival Curve showing association of XRCC1 Arg399Gln Genotype on Lung Cancer Overall Survival

20

40

60

80

10 0

Survival in months

Fig. 2 Kaplan Meier Survival Curve showing association of Stage on Lung Cancer Overall Survival

Discussion a median survival of 23 months (95% CI = 19.34–26.66). Patients with the XPD 751 AC and CC genotypes had a median survival of 20 (95% CI = 15.19–24.81) and 14 months (95% CI = 2.77–25.23), respectively (log rank P = 0.19). When both SNPs of XRCC1 and XPD were analysed in combination, carriers of XRCC1 399 GA + AA/ XPD 751 AA (Median Survival-31 months), XRCC1 399 GA + AA/XPD 751 AA (Median Survival-23 months) and XRCC1 399 GA + AA/XPD 751 AC + CC (Median Survival-18 months) genotypes were associated with reduced overall survival (log rank P-value = 0.08) but was not signiWcant. We next investigated the association of clinicopathological features of lung cancer with survival. We included histology, tumour stage and metastasis. But survival diVerences in this study were most apparent in individuals with higher stage disease who had median survival of 14 months compared to 31 months in patients with stage I and II (log rank P-value = 0.002) (Fig. 2) and those who had metastasis who had a median survival of 15 months compared to non metastatic group (log rank Pvalue = 0.02). To Wnd out which of the genotypes and clinical factors were independent prognostic factors inXuencing prognosis, eVects of survival were assessed using cox proportional hazard model. In the multivariate analysis, the Hazard Ratio (with the XRCC1 399 GG group being the reference) for XRCC1 399 GA was 1.8 (P = 0.07) compared to 3.0 in the AA group (P = 0.005), the diVerence being signiWcant. On the contrary, the relative risk of dying (with the XPD 751 AA group being the reference) was 1.3 for those with the AC genotype (P = 0.29) and 2.45 for the CC group (P = 0.06), but statistically not signiWcant. Regarding the clinical factors, higher tumour stage came out to be signiWcant (HR = 2.1, P = 0.02).

Recent lung cancer studies have focused on identifying the eVects of SNPs in candidate genes, among which DNA repair genes are increasingly being studied. In our study, we assessed two common polymorphisms of the XRCC1 and XPD genes that may inXuence DNA repair capacity and their association with lung cancer risk and overall survival. The polymorphisms chosen for this study also have been shown to have functional signiWcance and may be responsible, in part, in the inter individual DNA damage repair variations in the general population and for a low DNA repair capacity phenotype characteristic of cancer patients (Helzlsouer et al. 1996; Wei and Spitz 1997). In the present study, signiWcantly higher odds ratios were noted for XRCC1 399 AA genotype (P = 0.007) compared to their normal counterparts. Carriers of the XRCC1 399 AA variant have been shown to have higher levels of DNA adducts (Lunn et al. 1999) and to be at greater risk for ionizing radiation sensitivity (Hu et al. 2001) and tobaccorelated DNA damage (Duell et al. 2000; Abdel-Rahman and El Zein 2000; Lei et al. 2002). The variant genotypes of XRCC1 399 GA and AA were more frequent in patients (Zienolddiny et al. 2006; Chacko et al. 2005).The XRCC1 399 AA variant genotype and risk for lung cancer has been reported previously by (Divine et al. 2001) who reported an association between the this variant and elevated risk for lung adenocarcinoma (OR = 2.5, 95% CI = 1.1–5.8) and another hospital based study (Park et al. 2002) found a further increased risk among squamous cell cases among Koreans (OR = 3.3, 95% CI = 1.2–9.2). However, in another study (Hu et al. 2005) no signiWcant association for the XRCC1 exonic variant 399 and lung cancer risk was obtained. Two studies in Chinese populations also found no

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650

signiWcant association between XRCC1 G399A polymorphism and lung cancer (Ratnasinghe et al. 2001; Chen et al. 2002). In Caucasian populations, some studies reported that the XRCC1 399 AA genotype signiWcantly associated with lung cancer risk (Divine et al. 2001; Zhou et al. 2003b). These Wndings provide evidence that this speciWc polymorphism of the XRCC1 gene is associated with reduced DNA repair eYciency. The G399A polymorphism occurs in a region of the XRCC1 gene that contains biologically important domains (the PARP binding and the BRCT domain), and these domains have homology with other DNA repairrelated genes (Masson et al. 1998).The protein encoded by XPD is involved in transcription-coupled NER and is an integral member of the basal transcription factor TFIIH complex (which is necessary for normal transcription initiation NER. The XPD 751 CC variant gene leads to conformational change in the coded protein at the domain of interaction between the XPD protein and its helicase activator, p44 protein, inside the TFIIH complex (Coin et al. 1998). SigniWcantly risk modulating eVect was noticed for XPD 751 AC genotype with an odds ratio of 1.8 (95% CI = 1.233–2.807, P = 0.003) compared to normal genotype in the present study. The A751C variant of XPD has been associated with increased risk of lung cancer in several other studies (Spitz et al. 2003; Hou et al. 2002; Zhou et al. 2002b; Ramachandran et al. 2006). The carriers of XPD 751 AC genotype were at 2.7-fold (95% CI = 1.12– 6.93) higher risk of lung cancer than carriers of the AA genotype in one study (Yin et al. 2006) in northeastern Chinese population. In another study (Xing et al. 2002) compared with those having the 751 AA genotype, subjects carrying at least one variant were at a borderline increased risk of SCC of the lung (OR = 1.5; 95% CI = 0.94–2.46). In Norwegian lung cancer population, there was a signiWcant association of XPD variants in modulating NSCLC risk (Zienolddiny et al. 2006). So also, A > C transversions, however, were marginally increased among patients with at least one XPD variant allele compared with patients who were wild-type homozygotes (Hou et al. 2003). These studies are all consistent with the reports indicating that heterozygosity is considered to carry the risk. However, contrasting results also exist as in one study (De Ruyck et al. 2007). From a genetic perspective, in most multifactorial diseases, single polymorphisms in single genes are unlikely to alter the expression or function of speciWc proteins to the extent of producing a pathological phenotype. It is more likely that the combined eVect of diVerent SNPs in a gene produce a change in expression or protein function. Since a combination of XRCC1 as well as XPD genes could be more important than single SNPs, we looked for the eVect of combined genotypes with regard to lung cancer risk. Compared to the XRCC1 399 GG/XPD 751 AA genotype

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which being the reference genotype, the other combined variants namely, XRCC1 399 GG/XPD 751 AC + CC (P = 0.03), XRCC1 399 GA + AA/XPD 751 AA (P = 0.04), XRCC1 399 GA + AA/XPD 751 AC+CC (P = 0.01), had signiWcantly higher odds ratios. Another study (Zhou et al. 2003a) found that the risk of lung cancer amongst nonsmokers increased progressively with the increase in the number of high-risk alleles of XRCC1 and XPD genes. Another study (Gao et al. 2006) reported in patients with the XRCC1 399 AA genotype, which results in a lower BER capacity were more likely to have p53 mutations, compared with patients with GG genotype. In addition, the p53 mutation frequency increased with an increasing number of combined genotypes associated with a lower DNA repair capacity XPD 751, and XRCC1 399. The XRCC1 codon 399 AA genotype was also associated with adenine to guanine p53 mutations in non-small cell lung cancer in one study (Casse et al. 2003). Regarding distributions of XRCC1 399 and XPD 751 genotypes and clinical characteristics of lung cancer patients, even though higher odds ratios were obtained there were no signiWcant associations with histopathologic variables like tumour histology, metastasis or tumour stage. Because of XRCC1 and XPD are all involved in the DNA repair pathway; we explored the single/combined genotypes as well as clinicopathological features of lung cancer patients on overall survival of the patients with lung carcinoma. Data of 170 patients were available for the survival study, of which there were 66 deaths. Median Survival of the patients was 10 months. For patients possessing XRCC1 399 GG, XRCC1 GA and XRCC1 AA genotypes, the median survival was 31, 21 and 14 months, respectively. In the multivariate Cox model, XRCC1 399 genotypes remained an independent prognostic factor. The Hazard Ratio (with the XRCC1 Arg/Arg group being the reference) for XRCC1 GA was 1.8 (P = 0.07) compared to 3.04 in the AA group (P = 0.005), the diVerence being signiWcant. Patients with XPD AA genotype had a median survival of 23 months, while AC and CC genotypes had a median survival of 20 and 14 months, respectively (log rank P-value = 0.19). On the contrary, the relative risk of dying with the XPD AA (reference) was 1.3, for those with the AC genotype (P = 0.29) and 2.4 for the CC group (P = 0.06), but statistically not signiWcant. We could observe that the patients carrying none of the adverse genotypes (XRCC1 399 GG, XPD 751 AA and XRCC1 399 GG/XPD 751 AA) had much better survival than those carrying variant alleles. We could observe that the XRCC1 399 AA variant genotype was associated with a signiWcantly decreased overall survival. In the BER genes, the variant alleles of XRCC1 399 were signiWcantly associated and poor survival (HR = 1.9; 95% CI = 1.00–3.72) (Wu et al. 2006). Another study (Smith et al. 2007) reported that carriers of the glutamine-encoding allele at codon 751 of the


XPD DNA repair gene were signiWcantly more likely to have a karyotype associated with a less favourable prognosis. Another study (Gurubhagavatula et al. 2004) demonstrated that the XPD and XRCC1 variant genotypes, both alone and in combination, are associated with decreased overall survival in their patient population. However, polymorphism of codons 751 in the XPD gene did not aVect patient survival in one study (Ryu et al. 2004). These observations demonstrated for the Wrst time the eVect of polymorphisms of NER and BER genes on lung cancer patient survival in Indian lung cancer patients. Many individual clinical features of the patient may come into play. The stage of disease at the time of diagnosis not only plays a key role in the selected treatment but also has a direct impact on the survival rate. In our study, higher tumour stage had a signiWcant eVect on the survival which is in accordance with one study (Ahrendt et al. 2003). Multivariate analysis showed that higher stage (Stage II and III) was overall an independent prognostic factor with a twofold increased risk of death. It may be possible that those individuals with higher stage disease already have too many genetic alterations during their tumour growth which reduces their survival period. No signiWcant association with tumour histology was observed in our study. Approximately 60% of SCLC and 30–40% of NSCLC patients present with higher stage metastatic disease. Although lung cancer can metastasize to virtually any organ site, the most common sites that are clinically apparent are the central nervous system, bones, liver and adrenal glands. Those metastatic patients too had signiWcantly shorter survival probabilities (log rank Pvalue = 0.02) when compared to patients who had no metastasis but in the multivariate cox regression model after adjusting for other covariates, the hazard ratio was not signiWcant. Hence the results showed that pathologic stage also plays a role in inXuencing the survival for patients with lung cancer. In brief, to our knowledge, this is the Wrst report of XRCC1 and XPD polymorphisms in relation to lung cancer overall survival in Indian population. Our results suggests that XRCC1 399 AA and XPD 751GA genotypes might be risk genotypes for lung cancer. In our patients, presence of XRCC1 399 AA genotype and higher tumour stage were independent prognostic factors aVecting patient survival. The limitations of our study are our sample size, due to that our ability to precisely measure the magnitude of the eVect modiWcation or interaction of the genes is limited. Because DNA repair is a complex process with overlapping substrate speciWcity, it takes many proteins of various DNA repair pathways acting in concert to maintain cell viability and genome integrity and larger studies with biological characterization of the variant in diverse ethnic populations are needed to validate our Wndings.

651 Acknowledgements We wish to thank all the participants for their contribution. We also thank the support provided by the staV members of various clinical Departments of Regional Cancer Centre, Trivandrum. This work was supported by the grants from Department of Science & Technology, Govt. of India and Indian Council of Medical Research, Govt. of India.

References Abdel-Rahman SZ, El Zein RA (2000) The 399Gln polymorphism in the DNA repair gene XRCC1 modulates the genotoxic response induced in human lymphocytes by the tobacco-speciWc nitrosamine NNK. Cancer Lett 159:63–71 Ahrendt AS, Hu Y, Buta M, McDermot P, Benoit N, Yang SC, Sidransky D (2003) P53 mutations and survival in stage I non small cell Lung Cancer: results of a prospective study. J Natl Cancer Inst 95:926–927 Benhamou S, Sarasin A (2000) Variability in nucleotide excision repair and cancer risk: a review. Mutat Res 462:149–158 Butkiewicz D, Rusin M, Enewold L, Shields PG, Chorazy M, Harris CC (2001) Genetic polymorphisms in DNA repair genes and risk of lung cancer. Carcinogenesis 22:593–597 Caldecott KW (2003) XRCC1 and DNA strand break repair. DNA Repair (Amst) 2:955–969 Casse C, Hu YC, Ahrendt SA (2003) The XRCC1 codon 399 Gln allele is associated with adenine to guanine p53 mutations in non-small cell lung cancer. Mutat Res 528:19–27 Chacko P, Rajan B, Joseph T, Mathew BS, Pillai MR (2005) Polymorphisms in DNA repair gene XRCC1 and increased genetic susceptibility to breast cancer. Breast Cancer Res Treat 89:15–21 Chen S, Tang D, Xue K, Xu L, Ma G, Hsu Y, Cho SS (2002) DNA repair gene XRCC1 and XPD polymorphisms and risk of lung cancer in a Chinese population. Carcinogenesis 23:1321–1325 Coin F, Marinoni JF, Rodolfo C, Fribourg S, Pedrini AM, Egly JM (1998) Mutations in the XPD helicase gene result in XP and TTD phenotypes, preventing interaction between XPD and the p44 subunit of TFIIH. Nat Genet 20:184–188 De Ruyck K, Szaumkessel M, De Rudder I, Dehoorne A, Vral A, Claes K, Velghe A, Van Meerbeeck J, Thierens H (2007) Polymorphisms in base-excision repair and nucleotide-excision repair genes in relation to lung cancer risk. Mutat Res 631:101–110 Divine KK, Gilliland FD, Crowell RE, Stidley CA, Bocklage TJ, Cook DL, Belinsky SA (2001) The XRCC1 399 glutamine allele is a risk factor for adenocarcinoma of the lung. Mutat Res 461:273– 278 Duell EJ, Wiencke JK, Cheng TJ, Varkonyi A, Zuo ZF, Ashok TD, Mark EJ, Wain JC, Christiani DC, Kelsey KT (2000) Polymorphisms in the DNA repair genes XRCC1 and ERCC2 and biomarkers of DNA damage in human blood mononuclear cells. Carcinogenesis 21:965–971 Gao WM, Romkes M, Siegfried JM, Luketich JD, Keohavong P (2006) Polymorphisms in DNA repair genes XPD and XRCC1 and p53 mutations in lung carcinomas of never-smokers. Mol Carcinog 45:828–832 Gurubhagavatula S, Liu G, Park S, Zhou W, Su L, Wain JC, Lynch TJ, Neuberg DS, Christiani DC (2004) XPD and XRCC1 genetic polymorphisms are prognostic factors in advanced non-small-cell lung cancer patients treated with platinum chemotherapy. J Clin Oncol 22:2594–2601 Helzlsouer KJ, Harris EL, Parshad R, Perry HR, Price FM, Sanford KK (1996) DNA repair proWciency: potential susceptiblity factor for breast cancer. J Natl Cancer Inst 88:754–755 Hoeijmakers JH, Egly JM, Vermeulen W (1996) TFIIH: a key component in multiple DNA transactions. Curr Opin Genet Dev 6:26–33

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652 Hou SM, Falt S, Angelini S, Yang K, Nyberg F, Lambert B, Hemminki K (2002) The XPD variant alleles are associated with increased aromatic DNA adduct level and lung cancer risk. Carcinogenesis 23:599–603 Hou SM, Ryk C, Kannio A, Angelini S, Falt S, Nyberg F, HusgafvelPursiainen K (2003) InXuence of common XPD and XRCC1 variant alleles on p53 mutations in lung tumors. Environ Mol Mutagen 41:37–42 Hu JJ, Smith TR, Miller MS (2001) Amino acid substitution variants of APE1 and XRCC1 genes associated with ionizing radiation sensitivity. Carcinogenesis 22:917–922 Hu Z, Ma H, Chen F, Wei Q, Shen H (2005) XRCC1 polymorphisms and cancer risk: a meta-analysis of 38 case-control studies. Cancer Epidemiol Biomarkers Prev 14:1810–1818 Kubota Y, Nash RA, Klungland A, Schar P, Barnes DE, Lindahl T (1996) Reconstitution of DNA base excision-repair with puriWed human proteins: interaction between DNA polymerase beta and the XRCC1 protein. EMBO J 15:6662–6670 Leadon SA, Cooper PK (1993) Preferential repair of ionizing radiation-induced damage in the transcribed strand of an active human gene is defective in Cockayne syndrome. Proc Natl Acad Sci USA 90:10499–10503 Lehmann AR (2001) The xeroderma pigmentosum group D (XPD) gene: one gene, two functions, three diseases. Genes Dev 15:15–23 Lei YC, Hwang SJ, Chang CC, Kuo HW, Luo JC, Chang MJ, Cheng TJ (2002) EVects on sister chromatid exchange frequency of polymorphisms in DNA repair gene XRCC1 in smokers. Mutat Res 519:93–101 Lunn RM, Langlois RG, Hsieh LL, Thompson CL, Bell DA (1999) XRCC1 polymorphisms: eVects on aXatoxin B1-DNA adducts and glycophorin A variant frequency. Cancer Res 59:2557–2561 Lunn RM, Helzlsouer KJ, Parshad R, Umbach DM, Harris EL, Sanford KK, Bell DA (2000) XPD polymorphisms: eVects on DNA repair proWciency. Carcinogenesis 21:551–555 Masson M, Niedergang C, Schreiber V, Muller S, Menissier-de Murcia J, de Murcia G (1998) XRCC1 is speciWcally associated with poly(ADP-ribose) polymerase and negatively regulates its activity following DNA damage. Mol Cell Biol 18:3563–3571 Park JY, Lee SY, Jeon HS, Bae NC, Chae SC, Joo S, Kim CH, Park JH, Kam S, Kim IS, Jung TH (2002) Polymorphism of the DNA repair gene XRCC1 and risk of primary lung cancer. Cancer Epidemiol Biomarkers Prev 11:23–27 Ramachandran S, Ramadas K, Hariharan R, Rejnish KR, Radhakrishna PM (2006) Single nucleotide polymorphisms of DNA repair genes XRCC1 and XPD and its molecular mapping in Indian oral cancer. Oral Oncol 42:350–362 Ratnasinghe D, Yao SX, Tangrea JA, Qiao YL, Andersen MR, Barrett MJ, GiVen CA, Erozan Y, Tockman MS, Taylor PR (2001) Polymorphisms of the DNA repair gene XRCC1 and lung cancer risk. Cancer Epidemiol Biomarkers Prev 10:119–123 Ryu JS, Hong YC, Han HS, Lee JE, Kim S, Park YM, Kim YC, Hwang TS (2004) Association between polymorphisms of ERCC1 and XPD and survival in non-small-cell lung cancer patients treated with cisplatin combination chemotherapy. Lung Cancer 44:311–316 Shall S, de Murcia G (2000) Poly(ADP-ribose) polymerase-1: what have we learned from the deWcient mouse model? Mutat Res 460:1–15

123

J Cancer Res Clin Oncol (2008) 134:645–652 Shen MR, Jones IM, Mohrenweiser H (1998) Nonconservative amino acid substitution variants exist at polymorphic frequency in DNA repair genes in healthy humans. Cancer Res 58:604–608 Smith AG, Worrillow LJ, Allan JM (2007) A common genetic variant in XPD associates with risk of 5q- and 7q-deleted acute myeloid leukemia. Blood 109:1233–1236 Spitz MR, Wu X, Wang Y, Wang LE, Shete S, Amos CI, Guo Z, Lei L, Mohrenweiser H, Wei Q (2001) Modulation of nucleotide excision repair capacity by XPD polymorphisms in lung cancer patients. Cancer Res 61:1354–1357 Spitz MR, Wei Q, Dong Q (2003) Genetic susceptibility to lung cancer: the role of DNA damage and repair. Cancer Epidemiol Biomarkers Prev 12:689–698 Sung P, Bailly V, Weber C, Thompson LH, Prakash L, Prakash S (1993) Human xeroderma pigmentosum group D gene encodes a DNA helicase. Nature 365:852–855 Tang D, Cho S, Rundle A (2002) Polymorphisms in the DNA repair enzyme XPD are associated with increased levels of PAH-DNA adducts in a case-control study of breast cancer. Breast Cancer Res Treat 75:159–166 Tomescu D, Kavanagh G, Ha T, Campbell H, Melton DW (2001) Nucleotide excision repair gene XPD polymorphisms and genetic predisposition to melanoma. Carcinogenesis 22:403–408 Wei Q, Spitz MR (1997) The role of DNA repair capacity in susceptibility to lung cancer: a review. Cancer Metastasis Rev 16:295– 307 Wu X, Gu J, Wu TT, Swisher SG, Liao Z, Correa AM, Liu J, Etzel CJ, Amos CI, Huang M, Chiang SS, Milas L, Hittelman WN, Ajani JA (2006) Genetic variations in radiation and chemotherapy drug action pathways predict clinical outcomes in esophageal cancer. J Clin Oncol 24:3789–3798 Xing D, Tan W, Wei Q, Lin D (2002) Polymorphisms of the DNA repair gene XPD and risk of lung cancer in a Chinese population. Lung Cancer 38:123–129 Yin J, Vogel U, Ma Y, Guo L, Wang H, Qi R (2006) Polymorphism of the DNA repair gene ERCC2 Lys751Gln and risk of lung cancer in a northeastern Chinese population. Cancer Genet Cytogenet 169:27–32 Zhou W, Liu G, Miller DP (2002a) Gene-environment interaction for the ERCC2 polymorphisms and cumulative cigarette smoking exposure in lung cancer. Cancer Res 62:1377–1381 Zhou W, Liu G, Miller DP, Thurston SW, Xu LL, Wain JC, Lynch TJ, Su L, Christiani DC (2002b) Gene-environment interaction for the ERCC2 polymorphisms and cumulative cigarette smoking exposure in lung cancer. Cancer Res 62:1377–1381 Zhou W, Liu G, Miller DP, Thurston SW, Xu LL, Wain JC, Lynch TG, Su L, Christiani DC (2003a) Polymorphisms in the DNA repair genes XRCC1 and ERCC2, smoking, and lung cancer risk. Cancer Epidemiol Biomarkers Prev 12:359–365 Zhou W, Liu G, Miller DP, Thurston SW, Xu LL, Wain JC, Lynch TJ, Su L, Christiani DC (2003b) Polymorphisms in the DNA repair genes XRCC1 and ERCC2, smoking, and lung cancer risk. Cancer Epidemiol Biomarkers Prev 12:359–365 Zienolddiny S, Campa D, Lind H, Ryberg D, Skaug V, Stangeland L, Phillips DH, Canzian F, Haugen A (2006) Polymorphisms of DNA repair genes and risk of non-small cell lung cancer. Carcinogenesis 27:560–567