Genetic Variants of NBS 1 Predict Clinical Outcome of Platinum-based Chemotherapy in Advanced Non-small Cell Lung Cancer in Chinese

Lung cancer is the most common cancer worldwide with approximately 1.5 million newly diagnosed cases and 1 million deaths annually (Le Chevalier, 2010). Nonsmall cell lung cancer (NSCLC) accounts for 75%-85% of lung cancer and most NSCLC patients are diagnosed in advanced stage (Spiro and Silvestri, 2005). Although platinum-based combination chemotherapy is the first-line treatment for advanced NSCLC patients, the response rate was only about 40% (Kelly et al., 2001; Schiller et al., 2002). The tumor, lymph node, metastasis (TNM) staging system of lung cancer is used widely for predicting prognosis (Naruke et al., 2001). However, differences in response rate and survival among NSCLC patients with the same stage and chemotherapy regimen suggest that other important factors, such as genetic background, may also affect the prognosis of individual patient. Therefore,


Introduction
Lung cancer is the most common cancer worldwide with approximately 1.5 million newly diagnosed cases and 1 million deaths annually (Le Chevalier, 2010).Nonsmall cell lung cancer (NSCLC) accounts for 75%-85% of lung cancer and most NSCLC patients are diagnosed in advanced stage (Spiro and Silvestri, 2005).Although platinum-based combination chemotherapy is the first-line treatment for advanced NSCLC patients, the response rate was only about 40% (Kelly et al., 2001;Schiller et al., 2002).The tumor, lymph node, metastasis (TNM) staging system of lung cancer is used widely for predicting prognosis (Naruke et al., 2001).However, differences in response rate and survival among NSCLC patients with the same stage and chemotherapy regimen suggest that other important factors, such as genetic background, may also affect the prognosis of individual patient.Therefore,

Genetic Variants of NBS1 Predict Clinical Outcome of Platinum-based Chemotherapy in Advanced Non-small Cell Lung Cancer in Chinese
Jia-Li Xu 1& , Ling-Min Hu 2& , Ming-De Huang 3& , Wan Zhao 1 , Yong-Mei Yin 1 , Zhi-Bin Hu 2 , Hong-Xia Ma 2 , Hong-Bing Shen 2 , Yong-Qian Shu 1 * great efforts have been made to evaluate the role of genetic polymorphisms in predicting therapeutic response and prognosis of NSCLC patients treated with chemotherapy.
Platinum-based drugs are cytotoxic through the formation of platinum-DNA cross-links and adducts, causing cell cycle arrest and ultimately apoptosis if not properly repaired (Siddik, 2003).The efficacy of each combination (platinum plus another agent) has been demonstrated to be similar by a series of trials in unselected patients with response rates of 30% to 40% (Scagliotti et al., 2002;Schiller et al., 2002;Ohe et al., 2007).It has been suggested that a major factor for the development of resistance to platinum-based drugs is the enhanced DNA repair capacity of tumor cells, which prevents the accumulation of lethal DNA damage from cytotoxic agents (Perez, 1998).Therefore, the differences in DNA repair capacity may play a role in the treatment response and prognosis of cancer patients after chemotherapy.In humans, DNA double-strand break (DSB) repair plays a critical role in maintaining genomic stability by two distinct mechanisms: nonhomologous end joining (NHEJ) and homologous recombination (HR) (Kobayashi et al., 2004).A protein complex MRN (consisting of MRE11, RAD50 and NBS1) has been identified as the most important component in both of these two DSB pathways (Maser et al., 1997).Among those, the NBS1 protein is the key regulator of the MRN complex, which recruits MRE11/RAD50 or retains them at the vicinity of DNA damage sites by direct binding to histone H2AX.Mutations in NBS1 significantly downregulate MRN complex expression and markedly disrupt its function, thus enhancing cisplatin-induced DNA damage and cytotoxicity (Tran et al., 2004;Araki et al., 2010).Studies in mice have indicated that heterozygous for NBS1-null mutations develop tumors in lung, liver, mammary gland and prostate (Dumon-Jones et al., 2003;Zhang et al., 2006).NBS1 overexpression has been observed in several smoking-related cancer sites including NSCLC, hepatoma, esophageal cancer and head and neck squamous cell carcinoma (HNSCC) (Chen, 2005;Yang, 2006).Furthermore, it was also correlated with recurrence/ metastasis of oral squamous cell carcinoma and the poor prognosis of HNSCC (Yang, 2006;Hsu et al., 2010).Thus, NBS1 may play an important role in the development and prognosis of human cancers.
The NBS1 gene encompasses 48797 bps of genomic sequence on chromosome 8q21 with about 48 single nucleotide polymorphisms (SNPs) according to the National Center for Biotechnology Information dbSNPs public databases (Zhang et al., 2006).Rs1805794 located in exon 5 of NBS1 is one of the most common polymorphisms and can induce an amino acid substitution from Glu to Gln.Studies have investigated the association between rs1805794 and risk of multiple cancers, such as lung, breast, bladder and nasopharyngeal carcinoma (Medina et al., 2003;Lan et al., 2005;Lu et al., 2006;Hsu et al., 2007;Stern et al., 2009;Zheng et al., 2011).However, only one study has explored the effect of this polymorphism on prognosis of human cancer and reported that it was not associated with the survival of esophagus or gastric adenocarcinoma after platinum-based neoadjuvant poly-chemotherapy (Ott et al., 2011).Besides, rs13312840 (T>C) is another potentially functional SNP in NBS1, which is located in the 5' near gene of NBS1 and may modify the expression levels of NBS1 gene by interfere its' binding with transcription factor GATA-1 (Merika and Orkin, 1993).Studies have showed that rs13312840 was significantly associated with the risk of breast cancer in European and Cypriot populations (Lu et al., 2006;Loizidou et al., 2010).However, no study has focused on the association between rs13312840 and cancer prognosis.Given the important role of NBS1 in DSB repair, we hypothesize that the two SNPs in NBS1 may affect the function of NBS1 and then have associations with treatment response and prognosis of advanced NSCLC patients with platinum-based chemotherapy.
To validate this hypothesis, we investigated the association between these two polymorphisms of NBS1 (rs1805794 and rs13312840) and clinical outcome of lung cancer in 147 advanced NSCLC patients treated with platinum-based chemotherapy.

Study populations
All patients in the study were recruited from the First Affiliated Hospital of Nanjing Medical University (Jiangsu, China) between January 2004 and September 2010.All cases were newly diagnosed, histopathologically confirmed and without prior history of other cancers or previous chemo-or radio-therapy.Only inoperable, advanced NSCLC (IIIB-IV) patients were included in the study to avoid the potential confounding effect from surgery and clinical stage.Patients were interviewed face-to-face to collect demographic data including age, sex and smoking status.Those who had a low smoking frequency (<1 cigarette per day) and duration (<1 year) in their lifetime were defined as non-smokers; otherwise, they were classified as smokers.Each patient donated 5-ml venous blood after written informed consent was obtained.Finally, a total of 147 advanced NSCLC patients with available blood sample were included in our study.Patients' response to platinum-based (cisplatin or carboplatin) regimen was assessed after the first two or three cycles and determined by the Response Evaluation Criteria in Solid Tumors (RECIST) criteria 1.1 (Eisenhauer et al., 2009).All responses were re-evaluated at least 4 weeks after initial assessment.For data analysis, complete response (CR) and partial response (PR) were combined as responders, and stable disease (SD) along with progressive disease (PD) were grouped as nonresponders.Follow-up was performed per three months from the time of enrollment till death or the latest followup.Progression-free survival (PFS) was defined as the time from first treatment to the date of disease progression (PD), death or last follow-up.Overall survival (OS) was calculated as the time between first dose and death or the last follows.

DNA collection and genotyping
Genomic DNA was extracted from a leukocyte pellet by traditional proteinase K digestion and followed by phenol-chloroform extraction and ethanol precipitation.SNPs (rs1805794 and rs13312840) in NBS1 were genotyped with TaqMan genotyping assays, using the ABI 7900 real-time PCR system (Applied Biosystems

Statistical analysis
Hardy-Weinberg equilibrium was assessed by a goodness-of-fit χ 2 test.Odds ratios (ORs) and their 95% confidential intervals (CIs) were calculated as a measure of difference in the response rate using logistic regression analysis (responders vs. non-responders).The Kaplan-Meier and log-rank tests were used to assess the differences in PFS and OS.Median survival time (MST) was calculated, and mean survival time was presented when the MST could not be obtained.Cox proportional hazards model was applied to assess the hazard ratios (HRs) for PFS and OS.The adjustment factors included age, gender, smoking status, histology and stage.The statistical analyses were performed using Statistical Analysis System software (version 9.1.3,SAS Institute, Cary, NC).All P-values were two-sided, and P-value <0.05 was considered statistically significant.
Overall, 53 (36.1%) patients demonstrated responders, 94 (63.9%) showed no-responders.The distributions of were shown in Table 4. Genotype frequencies are in agreement with those expected according to the Hardy-Weinberg equilibrium model (P = 0.062 and 0.277 for rs1805794 and rs13312840, respectively).However, neither of the two polymorphisms of NBS1 was significantly associated with treatment response.

Polymorphisms and survival
Log-rank tests showed that NSCLC patients carrying rs1805794 CC genotype had an improved PFS compared to those with GG genotype (16 vs. 8 months, P = 0.040).
Similarly, multivariate cox regression analysis revealed a significant effect of NBS1 rs1805794 on patients' PFS after the adjustment for age, gender, smoking status, histology type and clinical stage (CC/CG vs. GG: adjusted HR = 0.62, 95% CI: 0.39-0.99;CC vs. CG/GG: adjusted HR 0.56, 95% CI: 0.32-0.97)(Table 4, Figure 1).Additionally, the variant homozygous of rs13312840 with a small sample size also showed a significant association with PFS NSCLC (CC vs. TC/TT: HR = 95% CI: 1.53-428.39).While only one subject was rs13312840 CC.Ultimately, the results should be treated with caution.No such associations were detected between these two polymorphisms and OS of NSCLC.
The associations between NBS1 rs1805794 polymorphism and PFS of advanced NSCLC were further stratified by age, gender, smoking, histology and stage.As shown in Table 5, the association remained significant among subjects with squamous cell carcinoma (HR = 0.26, 95% CI: 0.09-0.79)and clinical stage IV (HR = 0.53, 95% CI: 0.29-0.97).No significant heterogeneity existed in every stratum.

Discussion
This study demonstrated that NBS1 rs1805794 and rs13312840 variant genotypes were significantly associated with prognosis especially PFS in NSCLC patients with platinum-based chemotherapy, indicating that genetic variants in NBS1 might play a role in modulating advanced NSCLC patients' prognosis.
The NBS1 protein has three known functional regions: the N-terminus (amino acids 1-183), a central region (amino acids 278-343), and the C-terminus (amino    (Kobayashi et al., 2004).Among those, the N-terminal region contains a fork-head associated (FHA) domain and two breast cancer carboxy-terminal (BRCT) domains.Rs1805794 is a missense polymorphism (Glu185Gln) located in the BRCT domain of NBS1 (Kobayashi, 2004), and such a domain facilitates NBS1 to interact with breast cancer type 1 susceptibility protein (BRCA1) and form a BRCA1-associated genome surveillance complex (BASC) which is responsible for recognition and repair of aberrant DNA (Wang et al., 2000;Futaki and Liu, the Glu185Gln may interfere with protein-protein interaction and affect DNA repair capacity (Tauchi, 2000).Rs1061302, high linkage disequilibrium (LD) with rs1805794 (D'=1, R2=1 in CHB), was located in the MRN complex binding site and considered to be associated with increased risk of smoking-related cancers including lung cancer by Park et al.SNPs in LD with rs1061302 may inadvertently affect proper binding of the MRN complex and thus alter its ability to repair or detect DNA DSB (Park et al., 2010).Thus, we hypothesized that the C allele of rs1805794 confers a low level of MRN complex compared to the wild-type.Taken together, it seems this C variant allele is associated with decreased DNA repair activity.DNA repair has been termed a double-edged sword, with the reason that although decreased DNA repair may increase the susceptibility of cancer, it might also simultaneously improve survival in patients already diagnosed with cancer, when treated with agents inducing DNA damage like cisplatin or carboplatin (Li et al., 2009;Wang et al., 2011).
Publications on NBS1 polymorphisms have mainly focused on rs1805794 and the risk of cancers including lung, breast, bladder, renal cell cancers, non-Hodgkin lymphoma and childhood acute leukemia (Medina et al., 2003;Lan et al., 2005;Zienolddiny, 2005;Landi et al., 2006;Ryk et al., 2006;Hsu et al., 2007;Choudhury et al., 2008;Margulis et al., 2008;Mosor et al., 2008;Schuetz et al., 2009;Stern et al., 2009;Park et al., 2010), but the results were not consistent.A recent meta-analysis on rs1805794 has shed light on a positive association between the variant genotype of rs1805794 and the risk of multiple cancers (MeiXia et al., 2009).However, few studies have investigated the relationship between NBS1 and cancer patients' prognosis (Yang, 2006;Hsu et al., 2010;Ott et al., 2011).The only one study enrolling 258 esophageal cancer and gastric adenocarcinoma patients who receiving platin/5-Fu/leucovorin-based poly-chemotherapy showed that rs1805794 in NBS1 was not associated with recurrence or OS of patients with any of these two cancers in European population (Ott et al., 2011).Interestingly, we found that the NBS1 rs1805794 variant genotypes were associated with a longer PFS and OS, although the latter is not statistically significant.Different ethnicities, tumor sites or clinical characteristics of patients may explain why we observed associations while the previous study did not.Our findings supported that the variant allele of rs1805794 may affect DNA repair activity and in turn, play a role in the prognosis of NSCLC patients treated with platinumbased chemotherapy.The exact mechanisms for such association still need to be explored by functional studies.
Rs13312840 is located in the 5' UTR of NBS1 gene which is the binding site of transcription factor GATA-1 (Merika and Orkin, 1993).However, few studies have reported the association of this SNP with cancer development except that two studies focused on its effect on breast cancer risk with inconsistent results (Lu et al., 2006;Loizidou et al., 2010).In our study, we found a significant association between the homozygous variant genotype and an unfavorable prognosis; however, low power may be induced by small sample size of variant genotype.Therefore, our findings should be interpreted with caution and larger sample studies are needed to further understand this SNP.
Although our study reported some new findings about polymorphisms of NBS1 in NSCLC patients with chemotherapy, several potential limitations should be taken into consideration.Firstly, we only included two common functional SNPs, which is far from comprehensive.The observed associations may result from other polymorphisms in LD with the studied ones.Secondly, our study lacked the related phenotypic and functional assays, which limited our inquiry into the functional consequence of these variants.Moreover, our sample size was relatively small and other larger studies would address the associations of more SNPs with chemotherapy response and prognosis in such a patient population.
In summary, our findings suggest that polymorphisms in NBS1 may be genetic markers for prognosis of advanced NSCLC patients receiving platinum-based chemotherapy, which need the further validation from larger patient cohorts and functional evaluations.

Table 2 . Patient Characteristics and Clinical Features
CI, confidence interval; MST, median survival time; PFS, progression-free survival; OS, overall survival; a P value for χ2 test; b Information of smoking status was available in 146 cases; c Other carcinomas include large cell, undifferentiated and mixed-cell carcinoma; d Mean survival time was provided when MST could not be calculated

Table 3 . Treatment Characteristics of the Enrolled Patients
Foster City, CA).The sequences of primers and probes were listed in Table1.SDS allelic discrimination software (version 2.3, provided by ABI) was used for analysis of genotyping results.All the genotyping assays were performed with two blank (water) controls for quality control in each 384-well format and more than 10% samples were randomly selected to repeat, yielding a 100% concordant.The success rates of genotyping for rs1805794 and rs13312840 were 95.9% and 98.6%, respectively.

Figure 1. Kaplan-Meier Plot of PFS in Relation to the NBS1 rs1805794 Genotypes.
HR, hazard ratio; CI,