Studies on genetic polymorphism in nucleotide excision repair genes in relation to lung cancer patients

Abstract

DNA repair genes safeguard the genomic integrity from the exogenous as well as endogenous assaults. Molecular alterations in the repair pathway genes may lead to improper repair and ultimately carcinogenesis. Objectives To evaluate the role of single nucleotide polymorphic variants of Nucleotide Excision Repair (NER) genes i.e. XPC (Lys939Gln, Ala499Val), XPA (A23G, G709A), XPD (Lys939Gln, Asp312Asn, Arg156Arg), XPG (Phe670Leu, Asp1104His) and XPF (673C>T, 11985A>G and Arg415Gln) with lung cancer susceptibility, overall survival and clinical response. Gene-gene and gene-environment interaction were also assessed to understand their association with lung cancer. Methodology Genotyping of genomic DNA was carried out using PCR-RFLP (polymerase chain reaction restriction fragment length polymorphism) for each polymorphic site under study. Total number of subjects genotyped for each polymorphic variant was 740 (370 cases, 370 controls). Following this association analysis was carried out using logistic regression to obtain adjusted odds ratio and significance. Data mining analysis was performed including both Multi-dimensionality reduction (MDR) and Classification and Regression tree (CART) analysis to find the possible interaction between interacting SNP-SNP and gene-gene. Overall survival analysis for 311 cases was performed using Kaplan Meier survival analysis and Cox-regression analysis which gave adjusted hazards ratio. Multivariate logistic regression analysis for 202 samples was also conducted to evaluate association of different polymorphic variants with clinical response.

Results XPC Lys939Gln possessed risk for lung cancer (OR: 2.30; p=0.0007). Lys939Gln polymorphism also showed higher risk for all sub-groups of lung cancer. For subjects with mutant genotype (CC) had a 4 and 3-fold increased risk for ADCC (p<0.0001) and SQCC (p=0.009). The mutant genotype (CC) for Lys939Gln also presented a higher risk of developing lung cancer in heavy smokers (OR: 3.71; p=0.005). Another XPC polymorphic variant i.e. Ala499Val showed a protective association towards lung cancer (OR: 0.25; p=0.003). The survival analysis suggested no significant association between the XPC variants and survival of lung cancer patients. Further, the study showed association of A23G polymorphism with mutant genotype (GG) suggesting a 2.5-fold elevated risk for the disease (p= 0.0007). For lung cancer histological subtypes: SQCC (p=0.01), ADCC (p=0.05) and SCLC patients (p=0.0007) an increased lung cancer risk was observed risk. Smokers with mutant genotype (GG) exhibited a high risk for the lung cancer when compared to wild genotype (AA) for A23G polymorphism (p=0.0002). In G709A polymorphism, the heterozygous genotype (GA) displayed a protective effect towards the disease for subjects who were smokers (p=0.001). No significant association was observed between XPA polymorphism and overall survival in both Kaplan-Meier analysis and Cox regression model. The study further revealed association of mutant genotype (CC) with an increased lung cancer risk (p=0.01) and also with its all histological subtypes i.e. ADCC (p=0.03), SQCC (p=0.008) and SCLC (p=0.02) for XPD Lys751Gln polymorphism. Smokers carrying the Lys751Gln variant allele were associated with 2-fold increased risk of lung cancer (p=0.01). XPD Lys751Gln was analyzed as the best one factor model using Multidimensionality reduction (MDR) analysis and was associated with increased risk for lung cancer (p=<0.0001). Classification and regression tree (CART) analysis depicted XPD Arg156Arg as the highest risk group associated towards lung cancer susceptibility. A subgroup analysis based on chemotherapeutic regimens suggested that patients administered Irinotecan–platinum, for XPD Lys751Gln mutant genotype (CC) was significantly related to better OS as compared to wild type genotype (AA) (25.43 vs 5.63, Log rank p=0.01). The Cox model revealed a significant effect (HR=0.21, 95%CI=0.08-0.57, p=0.002), whereas a poor survival was reported for subjects who were carrying the mutant genotype (AA) for XPD Asp312Asn polymorphism and treated with same regimen as above in comparison with wild type genotype (GG) (1.86 vs 7.28, Log rank p=0.01). Patients treated with Pemetrexed–platinum doublets and carrying the heterozygous genotype of Asp312Asn polymorphism had a poor survival (HR=2.01, 95%CI=1.14-3.56, p=0.01). XPG Phe670Leu was associated with protective effect in lung cancer and its histological subtypes. It was observed that 673 C>T polymorphism might be associated with increased lung cancer risk. 11985 A>G and Arg415Gln polymorphism does not show any association with lung cancer risk. The genetic variants of 673 C>T polymorphism were associated with a better survival in ADCC patients while 11985 A>G polymorphism was associated with poor survival in SCLC patients. Lung cancer patients treated with irinotecan cisplatin/irinotecan carboplatin regimen indicated their association with shorter survival time and high risk of death in 11985 A>G polymorphism. The inter-gene analysis showed that individuals carrying multiple copies of risk variants have decreased risk for lung cancer. MDR analysis suggested prevalence of gene-gene interaction as well as gene-environment interaction. XPG Phe670Leu was predicted as the highest risk factor. CART analysis did not confirm gene-gene interaction but gene-smoking interaction was observed for NER SNPs. The combinatorial studies suggested that there was no association observed between polymorphic combinations of NER genes with overall survival in lung cancer patients. When applied on histological subtypes, the polymorphic were associated with poor survival showing a hazardous effect in SCLC patients. Conclusion This preliminary study provides an insight into the potential of genetic differences in NER genes to modulate lung cancer susceptibility. XPC Lys939GLn and XPD Lys751Gln were certainly modifying risk of lung cancer in North Indian population. XPG Phe670Leu also acted as an important risk modifier of lung cancer. ERCC2 functional SNPs (Lys751Gln and Asp312Asn) independently affect the OS of patients treated with irinotecan cisplatin/ irinotecan carboplatin regimen and may act as an important predictive parameter in survival of lung cancer patients. This study in future can facilitate assessment of genetic variations of XPD/ERCC2 and help patients to make therapeutic decisions for individualized therapy in advanced lung cancer patients.