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Association of ERCC1 Gene Polymorphisms (rs3212986 and rs11615) With the Risk of Lung Cancer in a Population From Southeast Iran Abstract Background: Polymorphisms within the excision repair cross-complementation group 1 (ERCC1), an essential component of DNA repair mechanisms, have been associated with various malignancies. This study aimed to evaluate the association of the single-nucleotide polymorphisms (SNPs) rs3212986 and rs11615 within the ERCC1 gene in non-small cell lung cancer (NSCLC) patients. Study Design: A case-control study. Methods: Genomic DNA was extracted from the peripheral blood samples of 83 NSCLC patients and 119 healthy individuals. The genetic diversity of SNPs rs3212986 and rs11615 was determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The RFLP results were confirmed through sequencing. Results: The TT genotype of the rs11615 SNP was associated with a higher risk of NSCLC development (odds ratio: 3.900, 95% confidence interval: 0.603, 22.866, P=0.050). Furthermore, the AA genotype of rs3212986 was related to a higher risk of NSCLC development (OR: 2.531, 95% CI: 1.017, 6.300, P=0.046). A significant association was observed between smoking and lung cancer (OR: 3.072, 95% CI: 1.715, 5.503, P<0.001). Moreover, among non-smokers, there was an association between lung cancer risk and the AA (OR: 6.825, 95% CI: 1.722, 27.044, P=0.006) and AC (OR: 2.503, 95% CI: 0.977, 6.412, P=0.056) genotypes of rs3212986. However, no correlation was found between the genotypes of these SNPs and patients’ sensitivity to cisplatin and carboplatin (P ˃ 0.05). Conclusion: The rs11615-related TT genotype and the rs3212986-related AA genotype may be associated with a higher risk of lung cancer development. Background Lung cancer remains one of the most prevalent cancers and the leading cause of cancer-related death worldwide.1 The incidence of lung cancer is increasing, especially in developing countries. In 2018, approximately 2.09 million new cases of lung cancer were reported, resulting in around 1.76 million deaths.2 Lung cancer is primarily classified into non-small cell lung cancer (NSCLC) and small cell lung types, accounting for approximately 85.0% and 15.0% of all malignant lung cancer cases, respectively.3 NSCLC cases are diagnosed in approximately two-thirds of patients’ primary at advanced stages (stage III or IV), with a 5-year survival rate of less than 15% due to lower response rates to standard treatment options.4 Common treatments for lung cancer include surgery, radiation, chemotherapy, and immunotherapy. However, considering that NSCLC is frequently diagnosed late, platinum-based chemotherapy or chemoradiotherapy is often the only option for most patients.5 Inter-individual diversity in response to medication was observed in NSCLC patients with the same disease stage and treatment regimen, indicating that genetic factors may significantly influence treatment efficacy.6 Chemotherapy using platinum compounds induces DNA damage by generating DNA-platinum adducts that activate the cell’s DNA repair machinery.7,8 The main mechanisms to protect DNA from mutagens and repair damage caused by various endogenous and exogenous agents include nucleotide excision repair, double-strand break repair, and base excision repair. Importantly, the excision repair cross-complementation group 1 (ERCC1) gene encodes a specific endonuclease within the nucleotide excision repair system that catalyzes the DNA strand breaks at the 5’ end of the damaged site. DAN double-strand breaks and cross-linking damages are also repaired by the ERCC1-encoded endonuclease.4,9,10 Genetic mutations within DNA repair genes can alter the expression, structure, and function of associated proteins, serving as important prognostic indicators for malignancies.1 Several studies suggest that the presence of certain functional polymorphisms within the ERCC1 gene may decrease DNA repair capabilities, thereby increasing the risk of various malignancies.11-14 Several other studies have also pointed to an association between ERCC1 variants and clinical outcomes in NSCLC patients.15,16 Regarding the carcinogenic potential of ERCC1 polymorphisms, these changes might serve as useful prognostic biomarkers in lung cancer. To the best of our knowledge, there is no report on the associations between polymorphisms of the ERCC1 gene and lung cancer in Iranian populations. Therefore, the current study has focused on the association of single-nucleotide polymorphisms (SNPs) rs3212986 and rs11615 within the ERCC1 gene with NSCLC in a southeastern Iranian population. Methods Study subjects In this case-control study, the sample size was determined by G*Power software, version 3.1.9.2. Considering the alpha error of 5%, test power of 80%, effect size of 0.218, and the degree of freedom of 2, a sample size of 204 was selected for this research.17,18 This study involved 83 NSCLC patients and 119 healthy subjects. NSCLC patients were recruited among those referred to the Javad Al-Aeme Clinic of Kerman University of Medical Sciences from May 2020 to September 2021. NSCLC was diagnosed by a respiratory specialist based on lung examination. Patient inclusion criteria were histological or cytological confirmed diagnosis of NSCLC. The healthy individuals were selected from blood donors at a regional blood transfusion center in Kerman, and none had a history of respiratory disease or other related disorders. Based on the data in Table 1, there was no significant difference between the demographic features of the study groups. Approval for the study protocol was obtained from the Ethics Committee of Kerman University of Medical Sciences (code IR.KMU.REC.1399.072). Furthermore, all participants were recruited after signing a written informed consent form. Peripheral blood samples (5 mL) were collected from each participant to determine the polymorphisms. Table 1 Demographic and clinicopathological characteristics for malignant patients Characteristics Control (n=119) Case (n=83) P value Number Percent Number Percent Gender Men 83 69.74 64 77.10 0.354 Women 36 30.25 19 22.89 Smoking status 0.001 Smokers 38 31.93 49 59.03 Non-smokers 81 68.06 34 40.96 Tumor stage I - 0 0 II - 0 0 III - 26 31.32 IV - 57 68.67 Histology Adenocarcinoma - 52 62.65 Squamous cell carcinoma - 29 31.32 Large cell carcinoma - 2 2.40 Response to chemotherapy Complete response + partial response - 16 40 Stable disease + progressive disease - 24 60 Chemotherapy regimen All patients were treated with cisplatin- or carboplatin-based chemotherapy every 3 weeks. After a median follow-up of 15 months, responses to treatment were determined and defined as NSCLC patients with complete response (CR) and partial response (PR) to chemotherapy. No-responders were defined as stable disease (SD) and progressive disease (PD). Genomic DNA extraction Genomic DNA was isolated from peripheral blood using a salting-out technique. Spectrophotometry (Eppendorf, Germany) was used to determine DNA quantities and purity according to the measurements of the absorbance at 260 nm and 280 nm. Extracted DNA samples were stored at -20°C until use. Genotyping assay The SNPs rs11615 and rs3212986 in ERCC1 were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The PCR was performed as 1 μL of prepared DNA, 3 μL of the PCR buffer (10x), 0.3 μL of the Taq DNA polymerase (5 U/mM), 1.5 μL of MgCl2 (stock concentration 1.5 mM), 0.5 μL dNTPs (stock concentration of 10 mM), 2 μL of each primer (300 nmol), and distilled water up to 30 μL. The forward (5′-GGTGCAAGAAGAGGTGGAG-3′ and 5′-AAGAAGCAGAGTCAGGAAAGC-3′) and reverse (5′-TCAGATCCCCAGGAGTCC-3′ and 5′-ACCCCACTCTAGATTTACCCAG-3) primers were used to amplify the regions around the SNPs rs11615 and rs3212986, respectively. The amplification program was designed as an initial denaturation step (95 ˚C for 5 minutes), followed by 40 cycles of denaturation (94 °C for 15 seconds), annealing (60 °C for 25 seconds), and extension (72°C for 30 seconds). Additionally, a final extension step was performed (72 °C for 10 minutes) to complete the PCR. The amplified PCR products contain SNPs rs11615 and rs3212986 with lengths of 471 bp and 442 bp, respectively. Subsequently, PCR products (5 μL) related to SNPs rs11615 and rs3212986 were digested overnight (37 °C) in the presence of 0.75 unit/reaction of BsrDI and MBOII restriction endonuclease, respectively. Considering that the BsrDI enzyme has exclusively one restriction site in the SNP rs11615-related PCR product, thus the CC genotype has two DNA fragments of 184 bp and 319 bp, whereas the AA genotype has a single undigested DNA fragment (503 bp), and the heterozygous CA genotype has three DNA fragments (503 bp, 319 bp, and 184 bp). The MBOII enzyme has only one restriction site in the PCR product associated with the rs3212986 SNP; thus, the CC genotype has two DNA fragments of 184 bp and 319 bp. However, the AA genotype has a single undigested DNA fragment (503 bp), and the heterozygous CA genotype has three DNA fragments (503 bp, 319 bp, and 184 bp). After adding 2 μL of DNA safe stain (Kowsar Biotech Company, Iran) to enzyme-treated products, they were electrophoresed on a 3% agarose gel and evaluated with Chemi-Doc model XRS (Bio-Rad, USA; Figure 1). Finally, the sequencing was performed on 22 samples for each SNP to confirm the RFLP results. Partial DNA sequence results for SNPs rs11615 and rs3212986 are shown in Figure 2. Figure 1 Agarose gel electrophoresis of amplified PCR product of ERCC1 rs11615 (a) and ERCC1 rs321986 (b). Columns: (1) Blank, (2) Uncut PCR product, (3) Ladder, (4) TT (a) and AA (b) Genotype, (5) TC (a) and AC (b) Genotype, and (6) CC genotype. Note. PCR: Polymerase chain reaction. Figure 2 Partial DNA sequence results regarding SNPs rs11615 (a) and rs3212986 (b). Note. SNP: single-nucleotide polymorphism Statistical analysis The data were analyzed by statistical SPSS software (version 26, Chicago, IL, USA). Differences in variables were analyzed by using appropriate statistical tests, including logistic regression, independent sample t-test, and the chi-square (χ2). P values less than 0.05 were considered statistically significant. Results Patient characteristics The clinicopathological characteristics of the malignant patients are provided in Table 1. The study was performed on 83 advanced NSCLC patients, including 64 men and 19 women, with a mean ( ± standard deviation) age of 56.26 ( ± 1.48) years. Of the patients, 26 were at stage III, and 57 were at stage IV. Forty-nine of all NSCLC patients were smokers. Of the 40 patients evaluated for the treatment response, 24 (60%) showed SD + PD (non-responsive state) to chemotherapy, of whom 10 (25%) died (PD). Among 16 (40%) responders (CR + PR), 3 (7.5%) had a CR. The control group included 119 normal or healthy people (83 men and 36 women, with a mean [ ± SD] age of 54.87 [ ± 1.18] years). The age was not significantly different between case and control groups (P = 0.463). Genetic variation of ERCC1 rs11615 single-nucleotide polymorphism The genotypes and allele frequencies of the ERCC1-related rs11615 SNP in malignant and control groups are presented in Table 2. The frequencies of CC, TC, and TT genotypes at the rs11615 of the ERCC1 gene were 36.14%, 53.01%, and 10.840% in lung cancer patients, as well as 32.77%, 64.70%, and 2.52% in the control group. A significant difference was found between lung cancer patients and controls in the frequency of the CC, TC, and TT genotypes at the rs11615 SNP (P = 0.031). The difference in the frequency of T and C alleles between the malignant patients and controls was not significant. Considering the CC genotype as a criterion, it was found that the TT genotype was associated with a higher risk of lung cancer development (odds ratio: 3.9, 95% confidence interval: 0.603, 22.866; P = 0.055, Table 2). Table 2 Frequency of genotypes and alleles at SNP rs11615 in the ERCC1 gene in malignant patients and healthy control group SNP rs11615 Lung Cancer Healthy Group P value OR (95% CI) P value Number Percent Number Percent Genotypes 0.031 CC 30 36.14 39 32.77 Ref. TC 44 53.02 77 64.70 0.742 (0.406, 1.358) 0.334 TT 9 10.84 3 2.52 3.900 (0.603, 22.866) 0.055 Alleles 0.750 C 104 62.65 155 65.12 Ref. T 62 37.35 83 34.87 1.113 (0.737, 1.681) 0.610 Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio. Genetic variation of ERCC1 rs3212986 single-nucleotide polymorphism The genotypes and allele frequencies of the ERCC1-related rs3212986 SNP in malignant and control groups are listed in Table 3. The frequencies of CC, AC, and CC genotypes at the rs3212986 of the ERCC1 gene were 38.55%, 43.37%, and 60.24% in lung cancer patients, as well as 45.37%, 46.21%, and 8.40% in the control group. Differences in the frequencies of CC, AC, and CC genotypes between the malignant and control groups were not significant. Similarly, there was no significant difference in allele A and allele C frequencies between the malignant and control groups. Considering the CC genotype as a reference, it was revealed that the AA genotype was associated with a higher risk of lung cancer development (OR: 2.531, 95% CI: 1.017, 6.300; P= 0.046, Table 3). Table 3 Frequency of genotypes and alleles at SNP rs3212986 in the ERCC1 gene in malignant patients and healthy control group SNP rs3212986 Lung Cancer Healthy Group P value OR (95% CI) P value Number Percent Number Percent Genotypes 0.116 CC 32 38.56 54 45.37 Ref. AC 36 43.37 55 46.22 1.105 (0.602, 2.026) 0.748 AA 15 18.07 10 8.41 2.531 (1.017, 6.300) 0.046 Alleles 0.087 C 100 60.25 163 68.48 Ref. A 66 39.75 75 31.52 0.697 (0.461, 1.055) 0.088 Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio. Genetic variation of ERCC1 single-nucleotide polymorphisms and smoking A significant association was observed between smoking and lung cancer (OR: 3.072, 95% CI: 1.715, 5.503; P < 0.001). The relationship between genotype and the risk of lung cancer was further analyzed in the malignant and healthy control groups based on smoking status, and there were no significant differences in this regard (Table 4). Based on the results, there was an association between the risk of lung cancer and AA (OR: 6.825, 95% CI: 1.722, 27.044; P= 0.006) and AC (OR: 2.503, 95% CI: 0.977, 6.412; P= 0.056) genotypes of the non-smoking group (Table 4). Table 4 Frequency of Genotypes at SNPs rs11615 and rs3212986 in the ERCC1 Gene in Malignant Patients and Healthy Control Group Based on Smoking Status Genotypes Smokers Non-smokers Case Control OR (95% CI) P value Case Control OR (95% CI) P value rs11615 TT 5 1 4.062 (0.420, 39.257) 0.226 4 2 3.714 (0.603, 22.866) 0.157 TC 28 24 0.948 (0.381, 2.361) 0.909 16 53 0.561 (0.238, 1.321) 0.186 CC 16 13 Ref. 14 26 Ref. rs3212986 AA 8 5 1.001 (0.275, 3.634) 0.999 7 5 6.825 (1.722, 27.044) 0.006 AC 17 18 0.590 (0.234, 1.489) 0.264 19 37 2.503 (0.977, 6.412) 0.056 CC 24 15 Ref. 8 39 Ref. Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio. Genetic variation of ERCC1 single-nucleotide polymorphisms and sensitivity to cisplatin and carboplatin The frequencies of the genotypes of ERCC1-related SNPs (rs11615 and rs3212986) based on sensitivity to cisplatin and carboplatin are presented in Table 5. No correlation with the genotypes was found in these SNPs and sensitivity to cisplatin and carboplatin (P = 0.520 and P = 0.458, respectively) in malignant patients. Table 5 Frequency of genotypes at SNPs rs11615 and rs3212986 in the ERCC1 gene in malignant patients based on sensitivity to cisplatin and carboplatin Total Frequency Responders Non-responders OR (95% CI) P value PR+CR SD+PD Genotype (rs11615) Cisplatin TT 2 1 1 1.001 (0.041, 24.547) 0.999 TC 19 6 13 2.166 (0.334, 14.057) 0.418 CC 6 3 3 Ref. Carboplatin TT 3 2 1 0.375 (0.022, 6.348) 0.497 TC 3 1 2 1.500 (0.089, 25.392) 0.779 CC 7 3 4 Ref. Genotype (rs3212986) Cisplatin AA 5 3 2 0.667 (0.069, 6.409) 0.725 AC 14 3 11 3.667 (0.5557, 24.132) 0.177 CC 8 4 4 Ref. Carboplatin AA 4 1 3 3.000 (0.188, 47.963) 0.437 AC 3 2 1 0.500 (0.028, 8.952) 0.638 CC 6 3 3 Ref. Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio. CR: Complete response; PR: Partial response; SD: Stable disease; PD: Progressive disease. Discussion ERCC1 plays a fundamental role in DNA repair, and over 100 polymorphisms have been identified in the ERCC1 gene that can influence the structure and function of the encoded protein.19 In this case-control study, the rs11615-related TT genotype and the s3212986-related AA genotype of the ERCC1 gene were associated with a higher risk of lung cancer development in Iranian patients, with advanced NSCLC cases who have been treated with platinum-based chemotherapy. Furthermore, the relationship between the rs11615 SNP genotype and lung cancer risk was analyzed based on smoking status, revealing an association between lung cancer risk and AA and AC genotypes in non-smokers. Previous studies reported a correlation between ERCC1 polymorphisms and NSCLC susceptibility, which supports our results. For instance, Yu et al showed that NSCLC patients with the rs3212986 AA genotype in the ERCC1 gene have an increased risk of lung cancer, particularly among smokers.20 Gao also demonstrated that ERCC1 genotype rs11615 TT and genotype rs3212986 AA contribute to increased mortality risk from NSCLC. Additionally, a study involving Chinese patients indicated that the presence of the AA genotype at ERCC1 rs3213986 was associated with an increased risk of lung cancer, particularly in smokers.21 Other studies have suggested the association between various ERCC1 genotypes and the risk of lung cancer. Chaszczewska-Markowska et al confirmed the role of the rs11615 T allele and the rs3212986 GG genotype in NSCLC susceptibility.1 Likewise, two additional studies highlighted the predictive significance of the ERCC1 rs3212986 polymorphism in NSCLC patients.19,22 There is also evidence that certain SNPs within the ERCC1 gene can lead to decreased levels of ERCC1 mRNA and protein, which potentially impair cellular repair ability and ultimately promote cancer development.23-26 Moreover, several studies have confirmed a relationship between ERCC1 gene polymorphisms and NSCLC treatment efficacy.10,11,15,27,28 The results of a studyin Chinese patients revealed that individuals with the rs11615 TT genotype and the T allele or the rs3212986 AA genotype and the A allele had significantly lower response rates to treatment. As a result, these SNPs could serve as predictors of treatment response and may be associated with increased mortality in patients with advanced NSCLC.21 It has been suggested that rs11615 (ERCC1) and rs3738948 (ERCC3) can influence the efficacy of platinum-based chemotherapy in NSCLC patients.29 On the other hand, some studies have failed to find a significant relationship between ERCC1 gene polymorphisms and treatment efficacy in NSCLC patients receiving cisplatin-based chemotherapy. The results of a study among Iranian patients with colorectal and gastric cancers showed no significant association between genetic variations in the ERCC1 rs11615 SNP and response to oxaliplatin-based chemotherapy.30 Consistent with these findings, no significant effect of ERCC1 polymorphisms on treatment response was observed in NSCLC patients receiving cisplatin-based chemotherapy. These contradictory results may be due to differences in population, case selection, and study design. In our study, the relatively small number of NSCLC patients with homozygous genotypes may affect the power of statistical analysis to determine an association between ERCC1 polymorphisms and treatment response. Several limitations of the current study require consideration. First, the relatively small sample size, especially of lung cancer cases, restricted the statistical power for subgroup analysis; thus, the results should be confirmed with studies of larger populations. Second, this study only evaluated the risk of lung cancer, but investigating prognostic potential, especially by measuring survival parameters, could be valuable. Finally, further experiments are needed to elucidate the specific contribution of ERCC1 polymorphisms to the susceptibility to lung cancer susceptibility as well as to reveal other functional SNPs involved in DNA repair pathways. Highlights ERCC1 rs11615 and rs3212986 polymorphisms are associated with a higher risk of lung cancer. SNPs rs11615 and rs3212986 may serve as useful predictive markers to identify individuals at increased risk of lung cancer. The TT genotype of the rs11615 SNP was associated with a higher risk of NSCLC development. Conclusion In summary, the findings revealed that the ERCC1 rs11615 and rs3212986 polymorphisms are associated with a higher risk of lung cancer. These SNPs may serve as useful predictive markers to identify individuals at increased risk of lung cancer. Authors’ Contribution Conceptualization: Yaser Masoumi-Ardakani, Abdollah Jafarzaheh, Behjat Kalantari Khandani. Data curation: Ali Khalouei, Arezu Khosravi Mashizi, Mohammadreza Zangouey, Mohammad Mehdi Yaghoobi, Beydolah Shahouzehi. Formal analysis: Mohammadreza Zangouey, Ali Khalouei. Funding acquisition: Yaser Masoumi-Ardakani. Investigation: Ali Khalouei, Yaser Masoumi-Ardakani. Methodology: Ali Khalouei, Arezu Khosravi Mashizi, Mohammad Mehdi Yaghoobi, Beydolah Shahouzehi. Project administration: Yaser Masoumi-Ardakani. Resources: Farnaz Sedghy, Behjat Kalantari Khandani. Software: Ali Khalouei, Mohammadreza Zangouey. Supervision: Yaser Masoumi-Ardakani. Validation: Yaser Masoumi-Ardakani, Farnaz Sedghy, Arezu Khosravi Mashizi, Mohammad Mehdi Yaghoobi. Visualization: Ali Khalouei, Arezu Khosravi Mashizi. Writing–original draft: Yaser Masoumi-Ardakani, Abdollah Jafarzaheh, Behjat Kalantari Khandani, Farnaz Sedghy, Arezu Khosravi Mashizi, Mohammad Mehdi Yaghoobi,Mohammadreza Zangouey, Beydolah Shahouzehi. Writing–review & editing: Yaser Masoumi-Ardakani, Farnaz Sedghy. Competing Interests The authors declare that there is no conflict of interests. Ethical Approval The current study was approved by the Ethics Committee of Kerman University of Medical Sciences and Health Services (IR.KMU.REC.1399.072), and all procedures were in accordance with standards set by the Declaration of Helsinki (1964). Funding This study was financially supported by the Kerman Student Research Committee (Grant number: 98000685).

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article-title	Association of ERCC1 Gene Polymorphisms (rs3212986 and rs11615) With the Risk of Lung Cancer in a Population From Southeast Iran
abstract	Background: Polymorphisms within the excision repair cross-complementation group 1 (ERCC1), an essential component of DNA repair mechanisms, have been associated with various malignancies. This study aimed to evaluate the association of the single-nucleotide polymorphisms (SNPs) rs3212986 and rs11615 within the ERCC1 gene in non-small cell lung cancer (NSCLC) patients. Study Design: A case-control study. Methods: Genomic DNA was extracted from the peripheral blood samples of 83 NSCLC patients and 119 healthy individuals. The genetic diversity of SNPs rs3212986 and rs11615 was determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The RFLP results were confirmed through sequencing. Results: The TT genotype of the rs11615 SNP was associated with a higher risk of NSCLC development (odds ratio: 3.900, 95% confidence interval: 0.603, 22.866, P=0.050). Furthermore, the AA genotype of rs3212986 was related to a higher risk of NSCLC development (OR: 2.531, 95% CI: 1.017, 6.300, P=0.046). A significant association was observed between smoking and lung cancer (OR: 3.072, 95% CI: 1.715, 5.503, P<0.001). Moreover, among non-smokers, there was an association between lung cancer risk and the AA (OR: 6.825, 95% CI: 1.722, 27.044, P=0.006) and AC (OR: 2.503, 95% CI: 0.977, 6.412, P=0.056) genotypes of rs3212986. However, no correlation was found between the genotypes of these SNPs and patients’ sensitivity to cisplatin and carboplatin (P ˃ 0.05). Conclusion: The rs11615-related TT genotype and the rs3212986-related AA genotype may be associated with a higher risk of lung cancer development.
p	Background: Polymorphisms within the excision repair cross-complementation group 1 (ERCC1), an essential component of DNA repair mechanisms, have been associated with various malignancies. This study aimed to evaluate the association of the single-nucleotide polymorphisms (SNPs) rs3212986 and rs11615 within the ERCC1 gene in non-small cell lung cancer (NSCLC) patients.
p	Study Design: A case-control study.
p	Methods: Genomic DNA was extracted from the peripheral blood samples of 83 NSCLC patients and 119 healthy individuals. The genetic diversity of SNPs rs3212986 and rs11615 was determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The RFLP results were confirmed through sequencing.
p	Results: The TT genotype of the rs11615 SNP was associated with a higher risk of NSCLC development (odds ratio: 3.900, 95% confidence interval: 0.603, 22.866, P=0.050). Furthermore, the AA genotype of rs3212986 was related to a higher risk of NSCLC development (OR: 2.531, 95% CI: 1.017, 6.300, P=0.046). A significant association was observed between smoking and lung cancer (OR: 3.072, 95% CI: 1.715, 5.503, P<0.001). Moreover, among non-smokers, there was an association between lung cancer risk and the AA (OR: 6.825, 95% CI: 1.722, 27.044, P=0.006) and AC (OR: 2.503, 95% CI: 0.977, 6.412, P=0.056) genotypes of rs3212986. However, no correlation was found between the genotypes of these SNPs and patients’ sensitivity to cisplatin and carboplatin (P ˃ 0.05).
p	Conclusion: The rs11615-related TT genotype and the rs3212986-related AA genotype may be associated with a higher risk of lung cancer development.
body	Background Lung cancer remains one of the most prevalent cancers and the leading cause of cancer-related death worldwide.1 The incidence of lung cancer is increasing, especially in developing countries. In 2018, approximately 2.09 million new cases of lung cancer were reported, resulting in around 1.76 million deaths.2 Lung cancer is primarily classified into non-small cell lung cancer (NSCLC) and small cell lung types, accounting for approximately 85.0% and 15.0% of all malignant lung cancer cases, respectively.3 NSCLC cases are diagnosed in approximately two-thirds of patients’ primary at advanced stages (stage III or IV), with a 5-year survival rate of less than 15% due to lower response rates to standard treatment options.4 Common treatments for lung cancer include surgery, radiation, chemotherapy, and immunotherapy. However, considering that NSCLC is frequently diagnosed late, platinum-based chemotherapy or chemoradiotherapy is often the only option for most patients.5 Inter-individual diversity in response to medication was observed in NSCLC patients with the same disease stage and treatment regimen, indicating that genetic factors may significantly influence treatment efficacy.6 Chemotherapy using platinum compounds induces DNA damage by generating DNA-platinum adducts that activate the cell’s DNA repair machinery.7,8 The main mechanisms to protect DNA from mutagens and repair damage caused by various endogenous and exogenous agents include nucleotide excision repair, double-strand break repair, and base excision repair. Importantly, the excision repair cross-complementation group 1 (ERCC1) gene encodes a specific endonuclease within the nucleotide excision repair system that catalyzes the DNA strand breaks at the 5’ end of the damaged site. DAN double-strand breaks and cross-linking damages are also repaired by the ERCC1-encoded endonuclease.4,9,10 Genetic mutations within DNA repair genes can alter the expression, structure, and function of associated proteins, serving as important prognostic indicators for malignancies.1 Several studies suggest that the presence of certain functional polymorphisms within the ERCC1 gene may decrease DNA repair capabilities, thereby increasing the risk of various malignancies.11-14 Several other studies have also pointed to an association between ERCC1 variants and clinical outcomes in NSCLC patients.15,16 Regarding the carcinogenic potential of ERCC1 polymorphisms, these changes might serve as useful prognostic biomarkers in lung cancer. To the best of our knowledge, there is no report on the associations between polymorphisms of the ERCC1 gene and lung cancer in Iranian populations. Therefore, the current study has focused on the association of single-nucleotide polymorphisms (SNPs) rs3212986 and rs11615 within the ERCC1 gene with NSCLC in a southeastern Iranian population. Methods Study subjects In this case-control study, the sample size was determined by G*Power software, version 3.1.9.2. Considering the alpha error of 5%, test power of 80%, effect size of 0.218, and the degree of freedom of 2, a sample size of 204 was selected for this research.17,18 This study involved 83 NSCLC patients and 119 healthy subjects. NSCLC patients were recruited among those referred to the Javad Al-Aeme Clinic of Kerman University of Medical Sciences from May 2020 to September 2021. NSCLC was diagnosed by a respiratory specialist based on lung examination. Patient inclusion criteria were histological or cytological confirmed diagnosis of NSCLC. The healthy individuals were selected from blood donors at a regional blood transfusion center in Kerman, and none had a history of respiratory disease or other related disorders. Based on the data in Table 1, there was no significant difference between the demographic features of the study groups. Approval for the study protocol was obtained from the Ethics Committee of Kerman University of Medical Sciences (code IR.KMU.REC.1399.072). Furthermore, all participants were recruited after signing a written informed consent form. Peripheral blood samples (5 mL) were collected from each participant to determine the polymorphisms. Table 1 Demographic and clinicopathological characteristics for malignant patients Characteristics Control (n=119) Case (n=83) P value Number Percent Number Percent Gender Men 83 69.74 64 77.10 0.354 Women 36 30.25 19 22.89 Smoking status 0.001 Smokers 38 31.93 49 59.03 Non-smokers 81 68.06 34 40.96 Tumor stage I - 0 0 II - 0 0 III - 26 31.32 IV - 57 68.67 Histology Adenocarcinoma - 52 62.65 Squamous cell carcinoma - 29 31.32 Large cell carcinoma - 2 2.40 Response to chemotherapy Complete response + partial response - 16 40 Stable disease + progressive disease - 24 60 Chemotherapy regimen All patients were treated with cisplatin- or carboplatin-based chemotherapy every 3 weeks. After a median follow-up of 15 months, responses to treatment were determined and defined as NSCLC patients with complete response (CR) and partial response (PR) to chemotherapy. No-responders were defined as stable disease (SD) and progressive disease (PD). Genomic DNA extraction Genomic DNA was isolated from peripheral blood using a salting-out technique. Spectrophotometry (Eppendorf, Germany) was used to determine DNA quantities and purity according to the measurements of the absorbance at 260 nm and 280 nm. Extracted DNA samples were stored at -20°C until use. Genotyping assay The SNPs rs11615 and rs3212986 in ERCC1 were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The PCR was performed as 1 μL of prepared DNA, 3 μL of the PCR buffer (10x), 0.3 μL of the Taq DNA polymerase (5 U/mM), 1.5 μL of MgCl2 (stock concentration 1.5 mM), 0.5 μL dNTPs (stock concentration of 10 mM), 2 μL of each primer (300 nmol), and distilled water up to 30 μL. The forward (5′-GGTGCAAGAAGAGGTGGAG-3′ and 5′-AAGAAGCAGAGTCAGGAAAGC-3′) and reverse (5′-TCAGATCCCCAGGAGTCC-3′ and 5′-ACCCCACTCTAGATTTACCCAG-3) primers were used to amplify the regions around the SNPs rs11615 and rs3212986, respectively. The amplification program was designed as an initial denaturation step (95 ˚C for 5 minutes), followed by 40 cycles of denaturation (94 °C for 15 seconds), annealing (60 °C for 25 seconds), and extension (72°C for 30 seconds). Additionally, a final extension step was performed (72 °C for 10 minutes) to complete the PCR. The amplified PCR products contain SNPs rs11615 and rs3212986 with lengths of 471 bp and 442 bp, respectively. Subsequently, PCR products (5 μL) related to SNPs rs11615 and rs3212986 were digested overnight (37 °C) in the presence of 0.75 unit/reaction of BsrDI and MBOII restriction endonuclease, respectively. Considering that the BsrDI enzyme has exclusively one restriction site in the SNP rs11615-related PCR product, thus the CC genotype has two DNA fragments of 184 bp and 319 bp, whereas the AA genotype has a single undigested DNA fragment (503 bp), and the heterozygous CA genotype has three DNA fragments (503 bp, 319 bp, and 184 bp). The MBOII enzyme has only one restriction site in the PCR product associated with the rs3212986 SNP; thus, the CC genotype has two DNA fragments of 184 bp and 319 bp. However, the AA genotype has a single undigested DNA fragment (503 bp), and the heterozygous CA genotype has three DNA fragments (503 bp, 319 bp, and 184 bp). After adding 2 μL of DNA safe stain (Kowsar Biotech Company, Iran) to enzyme-treated products, they were electrophoresed on a 3% agarose gel and evaluated with Chemi-Doc model XRS (Bio-Rad, USA; Figure 1). Finally, the sequencing was performed on 22 samples for each SNP to confirm the RFLP results. Partial DNA sequence results for SNPs rs11615 and rs3212986 are shown in Figure 2. Figure 1 Agarose gel electrophoresis of amplified PCR product of ERCC1 rs11615 (a) and ERCC1 rs321986 (b). Columns: (1) Blank, (2) Uncut PCR product, (3) Ladder, (4) TT (a) and AA (b) Genotype, (5) TC (a) and AC (b) Genotype, and (6) CC genotype. Note. PCR: Polymerase chain reaction. Figure 2 Partial DNA sequence results regarding SNPs rs11615 (a) and rs3212986 (b). Note. SNP: single-nucleotide polymorphism Statistical analysis The data were analyzed by statistical SPSS software (version 26, Chicago, IL, USA). Differences in variables were analyzed by using appropriate statistical tests, including logistic regression, independent sample t-test, and the chi-square (χ2). P values less than 0.05 were considered statistically significant. Results Patient characteristics The clinicopathological characteristics of the malignant patients are provided in Table 1. The study was performed on 83 advanced NSCLC patients, including 64 men and 19 women, with a mean ( ± standard deviation) age of 56.26 ( ± 1.48) years. Of the patients, 26 were at stage III, and 57 were at stage IV. Forty-nine of all NSCLC patients were smokers. Of the 40 patients evaluated for the treatment response, 24 (60%) showed SD + PD (non-responsive state) to chemotherapy, of whom 10 (25%) died (PD). Among 16 (40%) responders (CR + PR), 3 (7.5%) had a CR. The control group included 119 normal or healthy people (83 men and 36 women, with a mean [ ± SD] age of 54.87 [ ± 1.18] years). The age was not significantly different between case and control groups (P = 0.463). Genetic variation of ERCC1 rs11615 single-nucleotide polymorphism The genotypes and allele frequencies of the ERCC1-related rs11615 SNP in malignant and control groups are presented in Table 2. The frequencies of CC, TC, and TT genotypes at the rs11615 of the ERCC1 gene were 36.14%, 53.01%, and 10.840% in lung cancer patients, as well as 32.77%, 64.70%, and 2.52% in the control group. A significant difference was found between lung cancer patients and controls in the frequency of the CC, TC, and TT genotypes at the rs11615 SNP (P = 0.031). The difference in the frequency of T and C alleles between the malignant patients and controls was not significant. Considering the CC genotype as a criterion, it was found that the TT genotype was associated with a higher risk of lung cancer development (odds ratio: 3.9, 95% confidence interval: 0.603, 22.866; P = 0.055, Table 2). Table 2 Frequency of genotypes and alleles at SNP rs11615 in the ERCC1 gene in malignant patients and healthy control group SNP rs11615 Lung Cancer Healthy Group P value OR (95% CI) P value Number Percent Number Percent Genotypes 0.031 CC 30 36.14 39 32.77 Ref. TC 44 53.02 77 64.70 0.742 (0.406, 1.358) 0.334 TT 9 10.84 3 2.52 3.900 (0.603, 22.866) 0.055 Alleles 0.750 C 104 62.65 155 65.12 Ref. T 62 37.35 83 34.87 1.113 (0.737, 1.681) 0.610 Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio. Genetic variation of ERCC1 rs3212986 single-nucleotide polymorphism The genotypes and allele frequencies of the ERCC1-related rs3212986 SNP in malignant and control groups are listed in Table 3. The frequencies of CC, AC, and CC genotypes at the rs3212986 of the ERCC1 gene were 38.55%, 43.37%, and 60.24% in lung cancer patients, as well as 45.37%, 46.21%, and 8.40% in the control group. Differences in the frequencies of CC, AC, and CC genotypes between the malignant and control groups were not significant. Similarly, there was no significant difference in allele A and allele C frequencies between the malignant and control groups. Considering the CC genotype as a reference, it was revealed that the AA genotype was associated with a higher risk of lung cancer development (OR: 2.531, 95% CI: 1.017, 6.300; P= 0.046, Table 3). Table 3 Frequency of genotypes and alleles at SNP rs3212986 in the ERCC1 gene in malignant patients and healthy control group SNP rs3212986 Lung Cancer Healthy Group P value OR (95% CI) P value Number Percent Number Percent Genotypes 0.116 CC 32 38.56 54 45.37 Ref. AC 36 43.37 55 46.22 1.105 (0.602, 2.026) 0.748 AA 15 18.07 10 8.41 2.531 (1.017, 6.300) 0.046 Alleles 0.087 C 100 60.25 163 68.48 Ref. A 66 39.75 75 31.52 0.697 (0.461, 1.055) 0.088 Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio. Genetic variation of ERCC1 single-nucleotide polymorphisms and smoking A significant association was observed between smoking and lung cancer (OR: 3.072, 95% CI: 1.715, 5.503; P < 0.001). The relationship between genotype and the risk of lung cancer was further analyzed in the malignant and healthy control groups based on smoking status, and there were no significant differences in this regard (Table 4). Based on the results, there was an association between the risk of lung cancer and AA (OR: 6.825, 95% CI: 1.722, 27.044; P= 0.006) and AC (OR: 2.503, 95% CI: 0.977, 6.412; P= 0.056) genotypes of the non-smoking group (Table 4). Table 4 Frequency of Genotypes at SNPs rs11615 and rs3212986 in the ERCC1 Gene in Malignant Patients and Healthy Control Group Based on Smoking Status Genotypes Smokers Non-smokers Case Control OR (95% CI) P value Case Control OR (95% CI) P value rs11615 TT 5 1 4.062 (0.420, 39.257) 0.226 4 2 3.714 (0.603, 22.866) 0.157 TC 28 24 0.948 (0.381, 2.361) 0.909 16 53 0.561 (0.238, 1.321) 0.186 CC 16 13 Ref. 14 26 Ref. rs3212986 AA 8 5 1.001 (0.275, 3.634) 0.999 7 5 6.825 (1.722, 27.044) 0.006 AC 17 18 0.590 (0.234, 1.489) 0.264 19 37 2.503 (0.977, 6.412) 0.056 CC 24 15 Ref. 8 39 Ref. Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio. Genetic variation of ERCC1 single-nucleotide polymorphisms and sensitivity to cisplatin and carboplatin The frequencies of the genotypes of ERCC1-related SNPs (rs11615 and rs3212986) based on sensitivity to cisplatin and carboplatin are presented in Table 5. No correlation with the genotypes was found in these SNPs and sensitivity to cisplatin and carboplatin (P = 0.520 and P = 0.458, respectively) in malignant patients. Table 5 Frequency of genotypes at SNPs rs11615 and rs3212986 in the ERCC1 gene in malignant patients based on sensitivity to cisplatin and carboplatin Total Frequency Responders Non-responders OR (95% CI) P value PR+CR SD+PD Genotype (rs11615) Cisplatin TT 2 1 1 1.001 (0.041, 24.547) 0.999 TC 19 6 13 2.166 (0.334, 14.057) 0.418 CC 6 3 3 Ref. Carboplatin TT 3 2 1 0.375 (0.022, 6.348) 0.497 TC 3 1 2 1.500 (0.089, 25.392) 0.779 CC 7 3 4 Ref. Genotype (rs3212986) Cisplatin AA 5 3 2 0.667 (0.069, 6.409) 0.725 AC 14 3 11 3.667 (0.5557, 24.132) 0.177 CC 8 4 4 Ref. Carboplatin AA 4 1 3 3.000 (0.188, 47.963) 0.437 AC 3 2 1 0.500 (0.028, 8.952) 0.638 CC 6 3 3 Ref. Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio. CR: Complete response; PR: Partial response; SD: Stable disease; PD: Progressive disease. Discussion ERCC1 plays a fundamental role in DNA repair, and over 100 polymorphisms have been identified in the ERCC1 gene that can influence the structure and function of the encoded protein.19 In this case-control study, the rs11615-related TT genotype and the s3212986-related AA genotype of the ERCC1 gene were associated with a higher risk of lung cancer development in Iranian patients, with advanced NSCLC cases who have been treated with platinum-based chemotherapy. Furthermore, the relationship between the rs11615 SNP genotype and lung cancer risk was analyzed based on smoking status, revealing an association between lung cancer risk and AA and AC genotypes in non-smokers. Previous studies reported a correlation between ERCC1 polymorphisms and NSCLC susceptibility, which supports our results. For instance, Yu et al showed that NSCLC patients with the rs3212986 AA genotype in the ERCC1 gene have an increased risk of lung cancer, particularly among smokers.20 Gao also demonstrated that ERCC1 genotype rs11615 TT and genotype rs3212986 AA contribute to increased mortality risk from NSCLC. Additionally, a study involving Chinese patients indicated that the presence of the AA genotype at ERCC1 rs3213986 was associated with an increased risk of lung cancer, particularly in smokers.21 Other studies have suggested the association between various ERCC1 genotypes and the risk of lung cancer. Chaszczewska-Markowska et al confirmed the role of the rs11615 T allele and the rs3212986 GG genotype in NSCLC susceptibility.1 Likewise, two additional studies highlighted the predictive significance of the ERCC1 rs3212986 polymorphism in NSCLC patients.19,22 There is also evidence that certain SNPs within the ERCC1 gene can lead to decreased levels of ERCC1 mRNA and protein, which potentially impair cellular repair ability and ultimately promote cancer development.23-26 Moreover, several studies have confirmed a relationship between ERCC1 gene polymorphisms and NSCLC treatment efficacy.10,11,15,27,28 The results of a studyin Chinese patients revealed that individuals with the rs11615 TT genotype and the T allele or the rs3212986 AA genotype and the A allele had significantly lower response rates to treatment. As a result, these SNPs could serve as predictors of treatment response and may be associated with increased mortality in patients with advanced NSCLC.21 It has been suggested that rs11615 (ERCC1) and rs3738948 (ERCC3) can influence the efficacy of platinum-based chemotherapy in NSCLC patients.29 On the other hand, some studies have failed to find a significant relationship between ERCC1 gene polymorphisms and treatment efficacy in NSCLC patients receiving cisplatin-based chemotherapy. The results of a study among Iranian patients with colorectal and gastric cancers showed no significant association between genetic variations in the ERCC1 rs11615 SNP and response to oxaliplatin-based chemotherapy.30 Consistent with these findings, no significant effect of ERCC1 polymorphisms on treatment response was observed in NSCLC patients receiving cisplatin-based chemotherapy. These contradictory results may be due to differences in population, case selection, and study design. In our study, the relatively small number of NSCLC patients with homozygous genotypes may affect the power of statistical analysis to determine an association between ERCC1 polymorphisms and treatment response. Several limitations of the current study require consideration. First, the relatively small sample size, especially of lung cancer cases, restricted the statistical power for subgroup analysis; thus, the results should be confirmed with studies of larger populations. Second, this study only evaluated the risk of lung cancer, but investigating prognostic potential, especially by measuring survival parameters, could be valuable. Finally, further experiments are needed to elucidate the specific contribution of ERCC1 polymorphisms to the susceptibility to lung cancer susceptibility as well as to reveal other functional SNPs involved in DNA repair pathways. Highlights ERCC1 rs11615 and rs3212986 polymorphisms are associated with a higher risk of lung cancer. SNPs rs11615 and rs3212986 may serve as useful predictive markers to identify individuals at increased risk of lung cancer. The TT genotype of the rs11615 SNP was associated with a higher risk of NSCLC development. Conclusion In summary, the findings revealed that the ERCC1 rs11615 and rs3212986 polymorphisms are associated with a higher risk of lung cancer. These SNPs may serve as useful predictive markers to identify individuals at increased risk of lung cancer. Authors’ Contribution Conceptualization: Yaser Masoumi-Ardakani, Abdollah Jafarzaheh, Behjat Kalantari Khandani. Data curation: Ali Khalouei, Arezu Khosravi Mashizi, Mohammadreza Zangouey, Mohammad Mehdi Yaghoobi, Beydolah Shahouzehi. Formal analysis: Mohammadreza Zangouey, Ali Khalouei. Funding acquisition: Yaser Masoumi-Ardakani. Investigation: Ali Khalouei, Yaser Masoumi-Ardakani. Methodology: Ali Khalouei, Arezu Khosravi Mashizi, Mohammad Mehdi Yaghoobi, Beydolah Shahouzehi. Project administration: Yaser Masoumi-Ardakani. Resources: Farnaz Sedghy, Behjat Kalantari Khandani. Software: Ali Khalouei, Mohammadreza Zangouey. Supervision: Yaser Masoumi-Ardakani. Validation: Yaser Masoumi-Ardakani, Farnaz Sedghy, Arezu Khosravi Mashizi, Mohammad Mehdi Yaghoobi. Visualization: Ali Khalouei, Arezu Khosravi Mashizi. Writing–original draft: Yaser Masoumi-Ardakani, Abdollah Jafarzaheh, Behjat Kalantari Khandani, Farnaz Sedghy, Arezu Khosravi Mashizi, Mohammad Mehdi Yaghoobi,Mohammadreza Zangouey, Beydolah Shahouzehi. Writing–review & editing: Yaser Masoumi-Ardakani, Farnaz Sedghy. Competing Interests The authors declare that there is no conflict of interests. Ethical Approval The current study was approved by the Ethics Committee of Kerman University of Medical Sciences and Health Services (IR.KMU.REC.1399.072), and all procedures were in accordance with standards set by the Declaration of Helsinki (1964). Funding This study was financially supported by the Kerman Student Research Committee (Grant number: 98000685).
sec	Background Lung cancer remains one of the most prevalent cancers and the leading cause of cancer-related death worldwide.1 The incidence of lung cancer is increasing, especially in developing countries. In 2018, approximately 2.09 million new cases of lung cancer were reported, resulting in around 1.76 million deaths.2 Lung cancer is primarily classified into non-small cell lung cancer (NSCLC) and small cell lung types, accounting for approximately 85.0% and 15.0% of all malignant lung cancer cases, respectively.3 NSCLC cases are diagnosed in approximately two-thirds of patients’ primary at advanced stages (stage III or IV), with a 5-year survival rate of less than 15% due to lower response rates to standard treatment options.4 Common treatments for lung cancer include surgery, radiation, chemotherapy, and immunotherapy. However, considering that NSCLC is frequently diagnosed late, platinum-based chemotherapy or chemoradiotherapy is often the only option for most patients.5 Inter-individual diversity in response to medication was observed in NSCLC patients with the same disease stage and treatment regimen, indicating that genetic factors may significantly influence treatment efficacy.6 Chemotherapy using platinum compounds induces DNA damage by generating DNA-platinum adducts that activate the cell’s DNA repair machinery.7,8 The main mechanisms to protect DNA from mutagens and repair damage caused by various endogenous and exogenous agents include nucleotide excision repair, double-strand break repair, and base excision repair. Importantly, the excision repair cross-complementation group 1 (ERCC1) gene encodes a specific endonuclease within the nucleotide excision repair system that catalyzes the DNA strand breaks at the 5’ end of the damaged site. DAN double-strand breaks and cross-linking damages are also repaired by the ERCC1-encoded endonuclease.4,9,10 Genetic mutations within DNA repair genes can alter the expression, structure, and function of associated proteins, serving as important prognostic indicators for malignancies.1 Several studies suggest that the presence of certain functional polymorphisms within the ERCC1 gene may decrease DNA repair capabilities, thereby increasing the risk of various malignancies.11-14 Several other studies have also pointed to an association between ERCC1 variants and clinical outcomes in NSCLC patients.15,16 Regarding the carcinogenic potential of ERCC1 polymorphisms, these changes might serve as useful prognostic biomarkers in lung cancer. To the best of our knowledge, there is no report on the associations between polymorphisms of the ERCC1 gene and lung cancer in Iranian populations. Therefore, the current study has focused on the association of single-nucleotide polymorphisms (SNPs) rs3212986 and rs11615 within the ERCC1 gene with NSCLC in a southeastern Iranian population.
title	Background
p	Lung cancer remains one of the most prevalent cancers and the leading cause of cancer-related death worldwide.1 The incidence of lung cancer is increasing, especially in developing countries. In 2018, approximately 2.09 million new cases of lung cancer were reported, resulting in around 1.76 million deaths.2 Lung cancer is primarily classified into non-small cell lung cancer (NSCLC) and small cell lung types, accounting for approximately 85.0% and 15.0% of all malignant lung cancer cases, respectively.3 NSCLC cases are diagnosed in approximately two-thirds of patients’ primary at advanced stages (stage III or IV), with a 5-year survival rate of less than 15% due to lower response rates to standard treatment options.4 Common treatments for lung cancer include surgery, radiation, chemotherapy, and immunotherapy. However, considering that NSCLC is frequently diagnosed late, platinum-based chemotherapy or chemoradiotherapy is often the only option for most patients.5 Inter-individual diversity in response to medication was observed in NSCLC patients with the same disease stage and treatment regimen, indicating that genetic factors may significantly influence treatment efficacy.6
p	Chemotherapy using platinum compounds induces DNA damage by generating DNA-platinum adducts that activate the cell’s DNA repair machinery.7,8 The main mechanisms to protect DNA from mutagens and repair damage caused by various endogenous and exogenous agents include nucleotide excision repair, double-strand break repair, and base excision repair. Importantly, the excision repair cross-complementation group 1 (ERCC1) gene encodes a specific endonuclease within the nucleotide excision repair system that catalyzes the DNA strand breaks at the 5’ end of the damaged site. DAN double-strand breaks and cross-linking damages are also repaired by the ERCC1-encoded endonuclease.4,9,10
p	Genetic mutations within DNA repair genes can alter the expression, structure, and function of associated proteins, serving as important prognostic indicators for malignancies.1 Several studies suggest that the presence of certain functional polymorphisms within the ERCC1 gene may decrease DNA repair capabilities, thereby increasing the risk of various malignancies.11-14 Several other studies have also pointed to an association between ERCC1 variants and clinical outcomes in NSCLC patients.15,16 Regarding the carcinogenic potential of ERCC1 polymorphisms, these changes might serve as useful prognostic biomarkers in lung cancer. To the best of our knowledge, there is no report on the associations between polymorphisms of the ERCC1 gene and lung cancer in Iranian populations. Therefore, the current study has focused on the association of single-nucleotide polymorphisms (SNPs) rs3212986 and rs11615 within the ERCC1 gene with NSCLC in a southeastern Iranian population.
sec	Methods Study subjects In this case-control study, the sample size was determined by G*Power software, version 3.1.9.2. Considering the alpha error of 5%, test power of 80%, effect size of 0.218, and the degree of freedom of 2, a sample size of 204 was selected for this research.17,18 This study involved 83 NSCLC patients and 119 healthy subjects. NSCLC patients were recruited among those referred to the Javad Al-Aeme Clinic of Kerman University of Medical Sciences from May 2020 to September 2021. NSCLC was diagnosed by a respiratory specialist based on lung examination. Patient inclusion criteria were histological or cytological confirmed diagnosis of NSCLC. The healthy individuals were selected from blood donors at a regional blood transfusion center in Kerman, and none had a history of respiratory disease or other related disorders. Based on the data in Table 1, there was no significant difference between the demographic features of the study groups. Approval for the study protocol was obtained from the Ethics Committee of Kerman University of Medical Sciences (code IR.KMU.REC.1399.072). Furthermore, all participants were recruited after signing a written informed consent form. Peripheral blood samples (5 mL) were collected from each participant to determine the polymorphisms. Table 1 Demographic and clinicopathological characteristics for malignant patients Characteristics Control (n=119) Case (n=83) P value Number Percent Number Percent Gender Men 83 69.74 64 77.10 0.354 Women 36 30.25 19 22.89 Smoking status 0.001 Smokers 38 31.93 49 59.03 Non-smokers 81 68.06 34 40.96 Tumor stage I - 0 0 II - 0 0 III - 26 31.32 IV - 57 68.67 Histology Adenocarcinoma - 52 62.65 Squamous cell carcinoma - 29 31.32 Large cell carcinoma - 2 2.40 Response to chemotherapy Complete response + partial response - 16 40 Stable disease + progressive disease - 24 60 Chemotherapy regimen All patients were treated with cisplatin- or carboplatin-based chemotherapy every 3 weeks. After a median follow-up of 15 months, responses to treatment were determined and defined as NSCLC patients with complete response (CR) and partial response (PR) to chemotherapy. No-responders were defined as stable disease (SD) and progressive disease (PD). Genomic DNA extraction Genomic DNA was isolated from peripheral blood using a salting-out technique. Spectrophotometry (Eppendorf, Germany) was used to determine DNA quantities and purity according to the measurements of the absorbance at 260 nm and 280 nm. Extracted DNA samples were stored at -20°C until use. Genotyping assay The SNPs rs11615 and rs3212986 in ERCC1 were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The PCR was performed as 1 μL of prepared DNA, 3 μL of the PCR buffer (10x), 0.3 μL of the Taq DNA polymerase (5 U/mM), 1.5 μL of MgCl2 (stock concentration 1.5 mM), 0.5 μL dNTPs (stock concentration of 10 mM), 2 μL of each primer (300 nmol), and distilled water up to 30 μL. The forward (5′-GGTGCAAGAAGAGGTGGAG-3′ and 5′-AAGAAGCAGAGTCAGGAAAGC-3′) and reverse (5′-TCAGATCCCCAGGAGTCC-3′ and 5′-ACCCCACTCTAGATTTACCCAG-3) primers were used to amplify the regions around the SNPs rs11615 and rs3212986, respectively. The amplification program was designed as an initial denaturation step (95 ˚C for 5 minutes), followed by 40 cycles of denaturation (94 °C for 15 seconds), annealing (60 °C for 25 seconds), and extension (72°C for 30 seconds). Additionally, a final extension step was performed (72 °C for 10 minutes) to complete the PCR. The amplified PCR products contain SNPs rs11615 and rs3212986 with lengths of 471 bp and 442 bp, respectively. Subsequently, PCR products (5 μL) related to SNPs rs11615 and rs3212986 were digested overnight (37 °C) in the presence of 0.75 unit/reaction of BsrDI and MBOII restriction endonuclease, respectively. Considering that the BsrDI enzyme has exclusively one restriction site in the SNP rs11615-related PCR product, thus the CC genotype has two DNA fragments of 184 bp and 319 bp, whereas the AA genotype has a single undigested DNA fragment (503 bp), and the heterozygous CA genotype has three DNA fragments (503 bp, 319 bp, and 184 bp). The MBOII enzyme has only one restriction site in the PCR product associated with the rs3212986 SNP; thus, the CC genotype has two DNA fragments of 184 bp and 319 bp. However, the AA genotype has a single undigested DNA fragment (503 bp), and the heterozygous CA genotype has three DNA fragments (503 bp, 319 bp, and 184 bp). After adding 2 μL of DNA safe stain (Kowsar Biotech Company, Iran) to enzyme-treated products, they were electrophoresed on a 3% agarose gel and evaluated with Chemi-Doc model XRS (Bio-Rad, USA; Figure 1). Finally, the sequencing was performed on 22 samples for each SNP to confirm the RFLP results. Partial DNA sequence results for SNPs rs11615 and rs3212986 are shown in Figure 2. Figure 1 Agarose gel electrophoresis of amplified PCR product of ERCC1 rs11615 (a) and ERCC1 rs321986 (b). Columns: (1) Blank, (2) Uncut PCR product, (3) Ladder, (4) TT (a) and AA (b) Genotype, (5) TC (a) and AC (b) Genotype, and (6) CC genotype. Note. PCR: Polymerase chain reaction. Figure 2 Partial DNA sequence results regarding SNPs rs11615 (a) and rs3212986 (b). Note. SNP: single-nucleotide polymorphism Statistical analysis The data were analyzed by statistical SPSS software (version 26, Chicago, IL, USA). Differences in variables were analyzed by using appropriate statistical tests, including logistic regression, independent sample t-test, and the chi-square (χ2). P values less than 0.05 were considered statistically significant.
title	Methods
sec	Study subjects In this case-control study, the sample size was determined by G*Power software, version 3.1.9.2. Considering the alpha error of 5%, test power of 80%, effect size of 0.218, and the degree of freedom of 2, a sample size of 204 was selected for this research.17,18 This study involved 83 NSCLC patients and 119 healthy subjects. NSCLC patients were recruited among those referred to the Javad Al-Aeme Clinic of Kerman University of Medical Sciences from May 2020 to September 2021. NSCLC was diagnosed by a respiratory specialist based on lung examination. Patient inclusion criteria were histological or cytological confirmed diagnosis of NSCLC. The healthy individuals were selected from blood donors at a regional blood transfusion center in Kerman, and none had a history of respiratory disease or other related disorders. Based on the data in Table 1, there was no significant difference between the demographic features of the study groups. Approval for the study protocol was obtained from the Ethics Committee of Kerman University of Medical Sciences (code IR.KMU.REC.1399.072). Furthermore, all participants were recruited after signing a written informed consent form. Peripheral blood samples (5 mL) were collected from each participant to determine the polymorphisms. Table 1 Demographic and clinicopathological characteristics for malignant patients Characteristics Control (n=119) Case (n=83) P value Number Percent Number Percent Gender Men 83 69.74 64 77.10 0.354 Women 36 30.25 19 22.89 Smoking status 0.001 Smokers 38 31.93 49 59.03 Non-smokers 81 68.06 34 40.96 Tumor stage I - 0 0 II - 0 0 III - 26 31.32 IV - 57 68.67 Histology Adenocarcinoma - 52 62.65 Squamous cell carcinoma - 29 31.32 Large cell carcinoma - 2 2.40 Response to chemotherapy Complete response + partial response - 16 40 Stable disease + progressive disease - 24 60
title	Study subjects
p	In this case-control study, the sample size was determined by G*Power software, version 3.1.9.2. Considering the alpha error of 5%, test power of 80%, effect size of 0.218, and the degree of freedom of 2, a sample size of 204 was selected for this research.17,18 This study involved 83 NSCLC patients and 119 healthy subjects. NSCLC patients were recruited among those referred to the Javad Al-Aeme Clinic of Kerman University of Medical Sciences from May 2020 to September 2021. NSCLC was diagnosed by a respiratory specialist based on lung examination. Patient inclusion criteria were histological or cytological confirmed diagnosis of NSCLC. The healthy individuals were selected from blood donors at a regional blood transfusion center in Kerman, and none had a history of respiratory disease or other related disorders. Based on the data in Table 1, there was no significant difference between the demographic features of the study groups. Approval for the study protocol was obtained from the Ethics Committee of Kerman University of Medical Sciences (code IR.KMU.REC.1399.072). Furthermore, all participants were recruited after signing a written informed consent form. Peripheral blood samples (5 mL) were collected from each participant to determine the polymorphisms.
table-wrap	Table 1 Demographic and clinicopathological characteristics for malignant patients Characteristics Control (n=119) Case (n=83) P value Number Percent Number Percent Gender Men 83 69.74 64 77.10 0.354 Women 36 30.25 19 22.89 Smoking status 0.001 Smokers 38 31.93 49 59.03 Non-smokers 81 68.06 34 40.96 Tumor stage I - 0 0 II - 0 0 III - 26 31.32 IV - 57 68.67 Histology Adenocarcinoma - 52 62.65 Squamous cell carcinoma - 29 31.32 Large cell carcinoma - 2 2.40 Response to chemotherapy Complete response + partial response - 16 40 Stable disease + progressive disease - 24 60
label	Table 1
caption	Demographic and clinicopathological characteristics for malignant patients
title	Demographic and clinicopathological characteristics for malignant patients
table	Characteristics Control (n=119) Case (n=83) P value Number Percent Number Percent Gender Men 83 69.74 64 77.10 0.354 Women 36 30.25 19 22.89 Smoking status 0.001 Smokers 38 31.93 49 59.03 Non-smokers 81 68.06 34 40.96 Tumor stage I - 0 0 II - 0 0 III - 26 31.32 IV - 57 68.67 Histology Adenocarcinoma - 52 62.65 Squamous cell carcinoma - 29 31.32 Large cell carcinoma - 2 2.40 Response to chemotherapy Complete response + partial response - 16 40 Stable disease + progressive disease - 24 60
tr	Characteristics Control (n=119) Case (n=83) P value
td	Characteristics
td	Control (n=119)
td	Case (n=83)
td	P value
tr	Number Percent Number Percent
td	Number
td	Percent
td	Number
td	Percent
tr	Gender
td	Gender
tr	Men 83 69.74 64 77.10 0.354
td	Men
td	83
td	69.74
td	64
td	77.10
td	0.354
tr	Women 36 30.25 19 22.89
td	Women
td	36
td	30.25
td	19
td	22.89
tr	Smoking status 0.001
td	Smoking status
td	0.001
tr	Smokers 38 31.93 49 59.03
td	Smokers
td	38
td	31.93
td	49
td	59.03
tr	Non-smokers 81 68.06 34 40.96
td	Non-smokers
td	81
td	68.06
td	34
td	40.96
tr	Tumor stage
td	Tumor stage
tr	I - 0 0
td	I
td	-
td	0
td	0
tr	II - 0 0
td	II
td	-
td	0
td	0
tr	III - 26 31.32
td	III
td	-
td	26
td	31.32
tr	IV - 57 68.67
td	IV
td	-
td	57
td	68.67
tr	Histology
td	Histology
tr	Adenocarcinoma - 52 62.65
td	Adenocarcinoma
td	-
td	52
td	62.65
tr	Squamous cell carcinoma - 29 31.32
td	Squamous cell carcinoma
td	-
td	29
td	31.32
tr	Large cell carcinoma - 2 2.40
td	Large cell carcinoma
td	-
td	2
td	2.40
tr	Response to chemotherapy
td	Response to chemotherapy
tr	Complete response + partial response - 16 40
td	Complete response + partial response
td	-
td	16
td	40
tr	Stable disease + progressive disease - 24 60
td	Stable disease + progressive disease
td	-
td	24
td	60
sec	Chemotherapy regimen All patients were treated with cisplatin- or carboplatin-based chemotherapy every 3 weeks. After a median follow-up of 15 months, responses to treatment were determined and defined as NSCLC patients with complete response (CR) and partial response (PR) to chemotherapy. No-responders were defined as stable disease (SD) and progressive disease (PD).
title	Chemotherapy regimen
p	All patients were treated with cisplatin- or carboplatin-based chemotherapy every 3 weeks. After a median follow-up of 15 months, responses to treatment were determined and defined as NSCLC patients with complete response (CR) and partial response (PR) to chemotherapy. No-responders were defined as stable disease (SD) and progressive disease (PD).
sec	Genomic DNA extraction Genomic DNA was isolated from peripheral blood using a salting-out technique. Spectrophotometry (Eppendorf, Germany) was used to determine DNA quantities and purity according to the measurements of the absorbance at 260 nm and 280 nm. Extracted DNA samples were stored at -20°C until use.
title	Genomic DNA extraction
p	Genomic DNA was isolated from peripheral blood using a salting-out technique. Spectrophotometry (Eppendorf, Germany) was used to determine DNA quantities and purity according to the measurements of the absorbance at 260 nm and 280 nm. Extracted DNA samples were stored at -20°C until use.
sec	Genotyping assay The SNPs rs11615 and rs3212986 in ERCC1 were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The PCR was performed as 1 μL of prepared DNA, 3 μL of the PCR buffer (10x), 0.3 μL of the Taq DNA polymerase (5 U/mM), 1.5 μL of MgCl2 (stock concentration 1.5 mM), 0.5 μL dNTPs (stock concentration of 10 mM), 2 μL of each primer (300 nmol), and distilled water up to 30 μL. The forward (5′-GGTGCAAGAAGAGGTGGAG-3′ and 5′-AAGAAGCAGAGTCAGGAAAGC-3′) and reverse (5′-TCAGATCCCCAGGAGTCC-3′ and 5′-ACCCCACTCTAGATTTACCCAG-3) primers were used to amplify the regions around the SNPs rs11615 and rs3212986, respectively. The amplification program was designed as an initial denaturation step (95 ˚C for 5 minutes), followed by 40 cycles of denaturation (94 °C for 15 seconds), annealing (60 °C for 25 seconds), and extension (72°C for 30 seconds). Additionally, a final extension step was performed (72 °C for 10 minutes) to complete the PCR. The amplified PCR products contain SNPs rs11615 and rs3212986 with lengths of 471 bp and 442 bp, respectively. Subsequently, PCR products (5 μL) related to SNPs rs11615 and rs3212986 were digested overnight (37 °C) in the presence of 0.75 unit/reaction of BsrDI and MBOII restriction endonuclease, respectively. Considering that the BsrDI enzyme has exclusively one restriction site in the SNP rs11615-related PCR product, thus the CC genotype has two DNA fragments of 184 bp and 319 bp, whereas the AA genotype has a single undigested DNA fragment (503 bp), and the heterozygous CA genotype has three DNA fragments (503 bp, 319 bp, and 184 bp). The MBOII enzyme has only one restriction site in the PCR product associated with the rs3212986 SNP; thus, the CC genotype has two DNA fragments of 184 bp and 319 bp. However, the AA genotype has a single undigested DNA fragment (503 bp), and the heterozygous CA genotype has three DNA fragments (503 bp, 319 bp, and 184 bp). After adding 2 μL of DNA safe stain (Kowsar Biotech Company, Iran) to enzyme-treated products, they were electrophoresed on a 3% agarose gel and evaluated with Chemi-Doc model XRS (Bio-Rad, USA; Figure 1). Finally, the sequencing was performed on 22 samples for each SNP to confirm the RFLP results. Partial DNA sequence results for SNPs rs11615 and rs3212986 are shown in Figure 2. Figure 1 Agarose gel electrophoresis of amplified PCR product of ERCC1 rs11615 (a) and ERCC1 rs321986 (b). Columns: (1) Blank, (2) Uncut PCR product, (3) Ladder, (4) TT (a) and AA (b) Genotype, (5) TC (a) and AC (b) Genotype, and (6) CC genotype. Note. PCR: Polymerase chain reaction. Figure 2 Partial DNA sequence results regarding SNPs rs11615 (a) and rs3212986 (b). Note. SNP: single-nucleotide polymorphism
title	Genotyping assay
p	The SNPs rs11615 and rs3212986 in ERCC1 were determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The PCR was performed as 1 μL of prepared DNA, 3 μL of the PCR buffer (10x), 0.3 μL of the Taq DNA polymerase (5 U/mM), 1.5 μL of MgCl2 (stock concentration 1.5 mM), 0.5 μL dNTPs (stock concentration of 10 mM), 2 μL of each primer (300 nmol), and distilled water up to 30 μL. The forward (5′-GGTGCAAGAAGAGGTGGAG-3′ and 5′-AAGAAGCAGAGTCAGGAAAGC-3′) and reverse (5′-TCAGATCCCCAGGAGTCC-3′ and 5′-ACCCCACTCTAGATTTACCCAG-3) primers were used to amplify the regions around the SNPs rs11615 and rs3212986, respectively. The amplification program was designed as an initial denaturation step (95 ˚C for 5 minutes), followed by 40 cycles of denaturation (94 °C for 15 seconds), annealing (60 °C for 25 seconds), and extension (72°C for 30 seconds). Additionally, a final extension step was performed (72 °C for 10 minutes) to complete the PCR.
p	The amplified PCR products contain SNPs rs11615 and rs3212986 with lengths of 471 bp and 442 bp, respectively. Subsequently, PCR products (5 μL) related to SNPs rs11615 and rs3212986 were digested overnight (37 °C) in the presence of 0.75 unit/reaction of BsrDI and MBOII restriction endonuclease, respectively. Considering that the BsrDI enzyme has exclusively one restriction site in the SNP rs11615-related PCR product, thus the CC genotype has two DNA fragments of 184 bp and 319 bp, whereas the AA genotype has a single undigested DNA fragment (503 bp), and the heterozygous CA genotype has three DNA fragments (503 bp, 319 bp, and 184 bp). The MBOII enzyme has only one restriction site in the PCR product associated with the rs3212986 SNP; thus, the CC genotype has two DNA fragments of 184 bp and 319 bp. However, the AA genotype has a single undigested DNA fragment (503 bp), and the heterozygous CA genotype has three DNA fragments (503 bp, 319 bp, and 184 bp). After adding 2 μL of DNA safe stain (Kowsar Biotech Company, Iran) to enzyme-treated products, they were electrophoresed on a 3% agarose gel and evaluated with Chemi-Doc model XRS (Bio-Rad, USA; Figure 1). Finally, the sequencing was performed on 22 samples for each SNP to confirm the RFLP results. Partial DNA sequence results for SNPs rs11615 and rs3212986 are shown in Figure 2.
figure	Figure 1 Agarose gel electrophoresis of amplified PCR product of ERCC1 rs11615 (a) and ERCC1 rs321986 (b). Columns: (1) Blank, (2) Uncut PCR product, (3) Ladder, (4) TT (a) and AA (b) Genotype, (5) TC (a) and AC (b) Genotype, and (6) CC genotype. Note. PCR: Polymerase chain reaction.
label	Figure 1
p	Agarose gel electrophoresis of amplified PCR product of ERCC1 rs11615 (a) and ERCC1 rs321986 (b). Columns: (1) Blank, (2) Uncut PCR product, (3) Ladder, (4) TT (a) and AA (b) Genotype, (5) TC (a) and AC (b) Genotype, and (6) CC genotype. Note. PCR: Polymerase chain reaction.
figure	Figure 2 Partial DNA sequence results regarding SNPs rs11615 (a) and rs3212986 (b). Note. SNP: single-nucleotide polymorphism
label	Figure 2
p	Partial DNA sequence results regarding SNPs rs11615 (a) and rs3212986 (b). Note. SNP: single-nucleotide polymorphism
sec	Statistical analysis The data were analyzed by statistical SPSS software (version 26, Chicago, IL, USA). Differences in variables were analyzed by using appropriate statistical tests, including logistic regression, independent sample t-test, and the chi-square (χ2). P values less than 0.05 were considered statistically significant.
title	Statistical analysis
p	The data were analyzed by statistical SPSS software (version 26, Chicago, IL, USA). Differences in variables were analyzed by using appropriate statistical tests, including logistic regression, independent sample t-test, and the chi-square (χ2). P values less than 0.05 were considered statistically significant.
sec	Results Patient characteristics The clinicopathological characteristics of the malignant patients are provided in Table 1. The study was performed on 83 advanced NSCLC patients, including 64 men and 19 women, with a mean ( ± standard deviation) age of 56.26 ( ± 1.48) years. Of the patients, 26 were at stage III, and 57 were at stage IV. Forty-nine of all NSCLC patients were smokers. Of the 40 patients evaluated for the treatment response, 24 (60%) showed SD + PD (non-responsive state) to chemotherapy, of whom 10 (25%) died (PD). Among 16 (40%) responders (CR + PR), 3 (7.5%) had a CR. The control group included 119 normal or healthy people (83 men and 36 women, with a mean [ ± SD] age of 54.87 [ ± 1.18] years). The age was not significantly different between case and control groups (P = 0.463). Genetic variation of ERCC1 rs11615 single-nucleotide polymorphism The genotypes and allele frequencies of the ERCC1-related rs11615 SNP in malignant and control groups are presented in Table 2. The frequencies of CC, TC, and TT genotypes at the rs11615 of the ERCC1 gene were 36.14%, 53.01%, and 10.840% in lung cancer patients, as well as 32.77%, 64.70%, and 2.52% in the control group. A significant difference was found between lung cancer patients and controls in the frequency of the CC, TC, and TT genotypes at the rs11615 SNP (P = 0.031). The difference in the frequency of T and C alleles between the malignant patients and controls was not significant. Considering the CC genotype as a criterion, it was found that the TT genotype was associated with a higher risk of lung cancer development (odds ratio: 3.9, 95% confidence interval: 0.603, 22.866; P = 0.055, Table 2). Table 2 Frequency of genotypes and alleles at SNP rs11615 in the ERCC1 gene in malignant patients and healthy control group SNP rs11615 Lung Cancer Healthy Group P value OR (95% CI) P value Number Percent Number Percent Genotypes 0.031 CC 30 36.14 39 32.77 Ref. TC 44 53.02 77 64.70 0.742 (0.406, 1.358) 0.334 TT 9 10.84 3 2.52 3.900 (0.603, 22.866) 0.055 Alleles 0.750 C 104 62.65 155 65.12 Ref. T 62 37.35 83 34.87 1.113 (0.737, 1.681) 0.610 Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio. Genetic variation of ERCC1 rs3212986 single-nucleotide polymorphism The genotypes and allele frequencies of the ERCC1-related rs3212986 SNP in malignant and control groups are listed in Table 3. The frequencies of CC, AC, and CC genotypes at the rs3212986 of the ERCC1 gene were 38.55%, 43.37%, and 60.24% in lung cancer patients, as well as 45.37%, 46.21%, and 8.40% in the control group. Differences in the frequencies of CC, AC, and CC genotypes between the malignant and control groups were not significant. Similarly, there was no significant difference in allele A and allele C frequencies between the malignant and control groups. Considering the CC genotype as a reference, it was revealed that the AA genotype was associated with a higher risk of lung cancer development (OR: 2.531, 95% CI: 1.017, 6.300; P= 0.046, Table 3). Table 3 Frequency of genotypes and alleles at SNP rs3212986 in the ERCC1 gene in malignant patients and healthy control group SNP rs3212986 Lung Cancer Healthy Group P value OR (95% CI) P value Number Percent Number Percent Genotypes 0.116 CC 32 38.56 54 45.37 Ref. AC 36 43.37 55 46.22 1.105 (0.602, 2.026) 0.748 AA 15 18.07 10 8.41 2.531 (1.017, 6.300) 0.046 Alleles 0.087 C 100 60.25 163 68.48 Ref. A 66 39.75 75 31.52 0.697 (0.461, 1.055) 0.088 Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio. Genetic variation of ERCC1 single-nucleotide polymorphisms and smoking A significant association was observed between smoking and lung cancer (OR: 3.072, 95% CI: 1.715, 5.503; P < 0.001). The relationship between genotype and the risk of lung cancer was further analyzed in the malignant and healthy control groups based on smoking status, and there were no significant differences in this regard (Table 4). Based on the results, there was an association between the risk of lung cancer and AA (OR: 6.825, 95% CI: 1.722, 27.044; P= 0.006) and AC (OR: 2.503, 95% CI: 0.977, 6.412; P= 0.056) genotypes of the non-smoking group (Table 4). Table 4 Frequency of Genotypes at SNPs rs11615 and rs3212986 in the ERCC1 Gene in Malignant Patients and Healthy Control Group Based on Smoking Status Genotypes Smokers Non-smokers Case Control OR (95% CI) P value Case Control OR (95% CI) P value rs11615 TT 5 1 4.062 (0.420, 39.257) 0.226 4 2 3.714 (0.603, 22.866) 0.157 TC 28 24 0.948 (0.381, 2.361) 0.909 16 53 0.561 (0.238, 1.321) 0.186 CC 16 13 Ref. 14 26 Ref. rs3212986 AA 8 5 1.001 (0.275, 3.634) 0.999 7 5 6.825 (1.722, 27.044) 0.006 AC 17 18 0.590 (0.234, 1.489) 0.264 19 37 2.503 (0.977, 6.412) 0.056 CC 24 15 Ref. 8 39 Ref. Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio. Genetic variation of ERCC1 single-nucleotide polymorphisms and sensitivity to cisplatin and carboplatin The frequencies of the genotypes of ERCC1-related SNPs (rs11615 and rs3212986) based on sensitivity to cisplatin and carboplatin are presented in Table 5. No correlation with the genotypes was found in these SNPs and sensitivity to cisplatin and carboplatin (P = 0.520 and P = 0.458, respectively) in malignant patients. Table 5 Frequency of genotypes at SNPs rs11615 and rs3212986 in the ERCC1 gene in malignant patients based on sensitivity to cisplatin and carboplatin Total Frequency Responders Non-responders OR (95% CI) P value PR+CR SD+PD Genotype (rs11615) Cisplatin TT 2 1 1 1.001 (0.041, 24.547) 0.999 TC 19 6 13 2.166 (0.334, 14.057) 0.418 CC 6 3 3 Ref. Carboplatin TT 3 2 1 0.375 (0.022, 6.348) 0.497 TC 3 1 2 1.500 (0.089, 25.392) 0.779 CC 7 3 4 Ref. Genotype (rs3212986) Cisplatin AA 5 3 2 0.667 (0.069, 6.409) 0.725 AC 14 3 11 3.667 (0.5557, 24.132) 0.177 CC 8 4 4 Ref. Carboplatin AA 4 1 3 3.000 (0.188, 47.963) 0.437 AC 3 2 1 0.500 (0.028, 8.952) 0.638 CC 6 3 3 Ref. Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio. CR: Complete response; PR: Partial response; SD: Stable disease; PD: Progressive disease.
title	Results
sec	Patient characteristics The clinicopathological characteristics of the malignant patients are provided in Table 1. The study was performed on 83 advanced NSCLC patients, including 64 men and 19 women, with a mean ( ± standard deviation) age of 56.26 ( ± 1.48) years. Of the patients, 26 were at stage III, and 57 were at stage IV. Forty-nine of all NSCLC patients were smokers. Of the 40 patients evaluated for the treatment response, 24 (60%) showed SD + PD (non-responsive state) to chemotherapy, of whom 10 (25%) died (PD). Among 16 (40%) responders (CR + PR), 3 (7.5%) had a CR. The control group included 119 normal or healthy people (83 men and 36 women, with a mean [ ± SD] age of 54.87 [ ± 1.18] years). The age was not significantly different between case and control groups (P = 0.463).
title	Patient characteristics
p	The clinicopathological characteristics of the malignant patients are provided in Table 1. The study was performed on 83 advanced NSCLC patients, including 64 men and 19 women, with a mean ( ± standard deviation) age of 56.26 ( ± 1.48) years. Of the patients, 26 were at stage III, and 57 were at stage IV. Forty-nine of all NSCLC patients were smokers. Of the 40 patients evaluated for the treatment response, 24 (60%) showed SD + PD (non-responsive state) to chemotherapy, of whom 10 (25%) died (PD). Among 16 (40%) responders (CR + PR), 3 (7.5%) had a CR.
p	The control group included 119 normal or healthy people (83 men and 36 women, with a mean [ ± SD] age of 54.87 [ ± 1.18] years). The age was not significantly different between case and control groups (P = 0.463).
sec	Genetic variation of ERCC1 rs11615 single-nucleotide polymorphism The genotypes and allele frequencies of the ERCC1-related rs11615 SNP in malignant and control groups are presented in Table 2. The frequencies of CC, TC, and TT genotypes at the rs11615 of the ERCC1 gene were 36.14%, 53.01%, and 10.840% in lung cancer patients, as well as 32.77%, 64.70%, and 2.52% in the control group. A significant difference was found between lung cancer patients and controls in the frequency of the CC, TC, and TT genotypes at the rs11615 SNP (P = 0.031). The difference in the frequency of T and C alleles between the malignant patients and controls was not significant. Considering the CC genotype as a criterion, it was found that the TT genotype was associated with a higher risk of lung cancer development (odds ratio: 3.9, 95% confidence interval: 0.603, 22.866; P = 0.055, Table 2). Table 2 Frequency of genotypes and alleles at SNP rs11615 in the ERCC1 gene in malignant patients and healthy control group SNP rs11615 Lung Cancer Healthy Group P value OR (95% CI) P value Number Percent Number Percent Genotypes 0.031 CC 30 36.14 39 32.77 Ref. TC 44 53.02 77 64.70 0.742 (0.406, 1.358) 0.334 TT 9 10.84 3 2.52 3.900 (0.603, 22.866) 0.055 Alleles 0.750 C 104 62.65 155 65.12 Ref. T 62 37.35 83 34.87 1.113 (0.737, 1.681) 0.610 Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio.
title	Genetic variation of ERCC1 rs11615 single-nucleotide polymorphism
p	The genotypes and allele frequencies of the ERCC1-related rs11615 SNP in malignant and control groups are presented in Table 2. The frequencies of CC, TC, and TT genotypes at the rs11615 of the ERCC1 gene were 36.14%, 53.01%, and 10.840% in lung cancer patients, as well as 32.77%, 64.70%, and 2.52% in the control group. A significant difference was found between lung cancer patients and controls in the frequency of the CC, TC, and TT genotypes at the rs11615 SNP (P = 0.031). The difference in the frequency of T and C alleles between the malignant patients and controls was not significant. Considering the CC genotype as a criterion, it was found that the TT genotype was associated with a higher risk of lung cancer development (odds ratio: 3.9, 95% confidence interval: 0.603, 22.866; P = 0.055, Table 2).
table-wrap	Table 2 Frequency of genotypes and alleles at SNP rs11615 in the ERCC1 gene in malignant patients and healthy control group SNP rs11615 Lung Cancer Healthy Group P value OR (95% CI) P value Number Percent Number Percent Genotypes 0.031 CC 30 36.14 39 32.77 Ref. TC 44 53.02 77 64.70 0.742 (0.406, 1.358) 0.334 TT 9 10.84 3 2.52 3.900 (0.603, 22.866) 0.055 Alleles 0.750 C 104 62.65 155 65.12 Ref. T 62 37.35 83 34.87 1.113 (0.737, 1.681) 0.610 Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio.
label	Table 2
caption	Frequency of genotypes and alleles at SNP rs11615 in the ERCC1 gene in malignant patients and healthy control group
title	Frequency of genotypes and alleles at SNP rs11615 in the ERCC1 gene in malignant patients and healthy control group
table	SNP rs11615 Lung Cancer Healthy Group P value OR (95% CI) P value Number Percent Number Percent Genotypes 0.031 CC 30 36.14 39 32.77 Ref. TC 44 53.02 77 64.70 0.742 (0.406, 1.358) 0.334 TT 9 10.84 3 2.52 3.900 (0.603, 22.866) 0.055 Alleles 0.750 C 104 62.65 155 65.12 Ref. T 62 37.35 83 34.87 1.113 (0.737, 1.681) 0.610
tr	SNP rs11615 Lung Cancer Healthy Group P value OR (95% CI) P value
td	SNP rs11615
td	Lung Cancer
td	Healthy Group
td	P value
td	OR (95% CI)
td	P value
tr	Number Percent Number Percent
td	Number
td	Percent
td	Number
td	Percent
tr	Genotypes 0.031
td	Genotypes
td	0.031
tr	CC 30 36.14 39 32.77 Ref.
td	CC
td	30
td	36.14
td	39
td	32.77
td	Ref.
tr	TC 44 53.02 77 64.70 0.742 (0.406, 1.358) 0.334
td	TC
td	44
td	53.02
td	77
td	64.70
td	0.742 (0.406, 1.358)
td	0.334
tr	TT 9 10.84 3 2.52 3.900 (0.603, 22.866) 0.055
td	TT
td	9
td	10.84
td	3
td	2.52
td	3.900 (0.603, 22.866)
td	0.055
tr	Alleles 0.750
td	Alleles
td	0.750
tr	C 104 62.65 155 65.12 Ref.
td	C
td	104
td	62.65
td	155
td	65.12
td	Ref.
tr	T 62 37.35 83 34.87 1.113 (0.737, 1.681) 0.610
td	T
td	62
td	37.35
td	83
td	34.87
td	1.113 (0.737, 1.681)
td	0.610
table-wrap-foot	Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio.
footnote	Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio.
p	Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio.
sec	Genetic variation of ERCC1 rs3212986 single-nucleotide polymorphism The genotypes and allele frequencies of the ERCC1-related rs3212986 SNP in malignant and control groups are listed in Table 3. The frequencies of CC, AC, and CC genotypes at the rs3212986 of the ERCC1 gene were 38.55%, 43.37%, and 60.24% in lung cancer patients, as well as 45.37%, 46.21%, and 8.40% in the control group. Differences in the frequencies of CC, AC, and CC genotypes between the malignant and control groups were not significant. Similarly, there was no significant difference in allele A and allele C frequencies between the malignant and control groups. Considering the CC genotype as a reference, it was revealed that the AA genotype was associated with a higher risk of lung cancer development (OR: 2.531, 95% CI: 1.017, 6.300; P= 0.046, Table 3). Table 3 Frequency of genotypes and alleles at SNP rs3212986 in the ERCC1 gene in malignant patients and healthy control group SNP rs3212986 Lung Cancer Healthy Group P value OR (95% CI) P value Number Percent Number Percent Genotypes 0.116 CC 32 38.56 54 45.37 Ref. AC 36 43.37 55 46.22 1.105 (0.602, 2.026) 0.748 AA 15 18.07 10 8.41 2.531 (1.017, 6.300) 0.046 Alleles 0.087 C 100 60.25 163 68.48 Ref. A 66 39.75 75 31.52 0.697 (0.461, 1.055) 0.088 Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio.
title	Genetic variation of ERCC1 rs3212986 single-nucleotide polymorphism
p	The genotypes and allele frequencies of the ERCC1-related rs3212986 SNP in malignant and control groups are listed in Table 3. The frequencies of CC, AC, and CC genotypes at the rs3212986 of the ERCC1 gene were 38.55%, 43.37%, and 60.24% in lung cancer patients, as well as 45.37%, 46.21%, and 8.40% in the control group. Differences in the frequencies of CC, AC, and CC genotypes between the malignant and control groups were not significant. Similarly, there was no significant difference in allele A and allele C frequencies between the malignant and control groups. Considering the CC genotype as a reference, it was revealed that the AA genotype was associated with a higher risk of lung cancer development (OR: 2.531, 95% CI: 1.017, 6.300; P= 0.046, Table 3).
table-wrap	Table 3 Frequency of genotypes and alleles at SNP rs3212986 in the ERCC1 gene in malignant patients and healthy control group SNP rs3212986 Lung Cancer Healthy Group P value OR (95% CI) P value Number Percent Number Percent Genotypes 0.116 CC 32 38.56 54 45.37 Ref. AC 36 43.37 55 46.22 1.105 (0.602, 2.026) 0.748 AA 15 18.07 10 8.41 2.531 (1.017, 6.300) 0.046 Alleles 0.087 C 100 60.25 163 68.48 Ref. A 66 39.75 75 31.52 0.697 (0.461, 1.055) 0.088 Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio.
label	Table 3
caption	Frequency of genotypes and alleles at SNP rs3212986 in the ERCC1 gene in malignant patients and healthy control group
title	Frequency of genotypes and alleles at SNP rs3212986 in the ERCC1 gene in malignant patients and healthy control group
table	SNP rs3212986 Lung Cancer Healthy Group P value OR (95% CI) P value Number Percent Number Percent Genotypes 0.116 CC 32 38.56 54 45.37 Ref. AC 36 43.37 55 46.22 1.105 (0.602, 2.026) 0.748 AA 15 18.07 10 8.41 2.531 (1.017, 6.300) 0.046 Alleles 0.087 C 100 60.25 163 68.48 Ref. A 66 39.75 75 31.52 0.697 (0.461, 1.055) 0.088
tr	SNP rs3212986 Lung Cancer Healthy Group P value OR (95% CI) P value
td	SNP rs3212986
td	Lung Cancer
td	Healthy Group
td	P value
td	OR (95% CI)
td	P value
tr	Number Percent Number Percent
td	Number
td	Percent
td	Number
td	Percent
tr	Genotypes 0.116
td	Genotypes
td	0.116
tr	CC 32 38.56 54 45.37 Ref.
td	CC
td	32
td	38.56
td	54
td	45.37
td	Ref.
tr	AC 36 43.37 55 46.22 1.105 (0.602, 2.026) 0.748
td	AC
td	36
td	43.37
td	55
td	46.22
td	1.105 (0.602, 2.026)
td	0.748
tr	AA 15 18.07 10 8.41 2.531 (1.017, 6.300) 0.046
td	AA
td	15
td	18.07
td	10
td	8.41
td	2.531 (1.017, 6.300)
td	0.046
tr	Alleles 0.087
td	Alleles
td	0.087
tr	C 100 60.25 163 68.48 Ref.
td	C
td	100
td	60.25
td	163
td	68.48
td	Ref.
tr	A 66 39.75 75 31.52 0.697 (0.461, 1.055) 0.088
td	A
td	66
td	39.75
td	75
td	31.52
td	0.697 (0.461, 1.055)
td	0.088
table-wrap-foot	Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio.
footnote	Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio.
p	Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio.
sec	Genetic variation of ERCC1 single-nucleotide polymorphisms and smoking A significant association was observed between smoking and lung cancer (OR: 3.072, 95% CI: 1.715, 5.503; P < 0.001). The relationship between genotype and the risk of lung cancer was further analyzed in the malignant and healthy control groups based on smoking status, and there were no significant differences in this regard (Table 4). Based on the results, there was an association between the risk of lung cancer and AA (OR: 6.825, 95% CI: 1.722, 27.044; P= 0.006) and AC (OR: 2.503, 95% CI: 0.977, 6.412; P= 0.056) genotypes of the non-smoking group (Table 4). Table 4 Frequency of Genotypes at SNPs rs11615 and rs3212986 in the ERCC1 Gene in Malignant Patients and Healthy Control Group Based on Smoking Status Genotypes Smokers Non-smokers Case Control OR (95% CI) P value Case Control OR (95% CI) P value rs11615 TT 5 1 4.062 (0.420, 39.257) 0.226 4 2 3.714 (0.603, 22.866) 0.157 TC 28 24 0.948 (0.381, 2.361) 0.909 16 53 0.561 (0.238, 1.321) 0.186 CC 16 13 Ref. 14 26 Ref. rs3212986 AA 8 5 1.001 (0.275, 3.634) 0.999 7 5 6.825 (1.722, 27.044) 0.006 AC 17 18 0.590 (0.234, 1.489) 0.264 19 37 2.503 (0.977, 6.412) 0.056 CC 24 15 Ref. 8 39 Ref. Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio.
title	Genetic variation of ERCC1 single-nucleotide polymorphisms and smoking
p	A significant association was observed between smoking and lung cancer (OR: 3.072, 95% CI: 1.715, 5.503; P < 0.001). The relationship between genotype and the risk of lung cancer was further analyzed in the malignant and healthy control groups based on smoking status, and there were no significant differences in this regard (Table 4). Based on the results, there was an association between the risk of lung cancer and AA (OR: 6.825, 95% CI: 1.722, 27.044; P= 0.006) and AC (OR: 2.503, 95% CI: 0.977, 6.412; P= 0.056) genotypes of the non-smoking group (Table 4).
table-wrap	Table 4 Frequency of Genotypes at SNPs rs11615 and rs3212986 in the ERCC1 Gene in Malignant Patients and Healthy Control Group Based on Smoking Status Genotypes Smokers Non-smokers Case Control OR (95% CI) P value Case Control OR (95% CI) P value rs11615 TT 5 1 4.062 (0.420, 39.257) 0.226 4 2 3.714 (0.603, 22.866) 0.157 TC 28 24 0.948 (0.381, 2.361) 0.909 16 53 0.561 (0.238, 1.321) 0.186 CC 16 13 Ref. 14 26 Ref. rs3212986 AA 8 5 1.001 (0.275, 3.634) 0.999 7 5 6.825 (1.722, 27.044) 0.006 AC 17 18 0.590 (0.234, 1.489) 0.264 19 37 2.503 (0.977, 6.412) 0.056 CC 24 15 Ref. 8 39 Ref. Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio.
label	Table 4
caption	Frequency of Genotypes at SNPs rs11615 and rs3212986 in the ERCC1 Gene in Malignant Patients and Healthy Control Group Based on Smoking Status
title	Frequency of Genotypes at SNPs rs11615 and rs3212986 in the ERCC1 Gene in Malignant Patients and Healthy Control Group Based on Smoking Status
table	Genotypes Smokers Non-smokers Case Control OR (95% CI) P value Case Control OR (95% CI) P value rs11615 TT 5 1 4.062 (0.420, 39.257) 0.226 4 2 3.714 (0.603, 22.866) 0.157 TC 28 24 0.948 (0.381, 2.361) 0.909 16 53 0.561 (0.238, 1.321) 0.186 CC 16 13 Ref. 14 26 Ref. rs3212986 AA 8 5 1.001 (0.275, 3.634) 0.999 7 5 6.825 (1.722, 27.044) 0.006 AC 17 18 0.590 (0.234, 1.489) 0.264 19 37 2.503 (0.977, 6.412) 0.056 CC 24 15 Ref. 8 39 Ref.
tr	Genotypes Smokers Non-smokers
td	Genotypes
td	Smokers
td	Non-smokers
tr	Case Control OR (95% CI) P value Case Control OR (95% CI) P value
td	Case
td	Control
td	OR (95% CI)
td	P value
td	Case
td	Control
td	OR (95% CI)
td	P value
tr	rs11615
td	rs11615
tr	TT 5 1 4.062 (0.420, 39.257) 0.226 4 2 3.714 (0.603, 22.866) 0.157
td	TT
td	5
td	1
td	4.062 (0.420, 39.257)
td	0.226
td	4
td	2
td	3.714 (0.603, 22.866)
td	0.157
tr	TC 28 24 0.948 (0.381, 2.361) 0.909 16 53 0.561 (0.238, 1.321) 0.186
td	TC
td	28
td	24
td	0.948 (0.381, 2.361)
td	0.909
td	16
td	53
td	0.561 (0.238, 1.321)
td	0.186
tr	CC 16 13 Ref. 14 26 Ref.
td	CC
td	16
td	13
td	Ref.
td	14
td	26
td	Ref.
tr	rs3212986
td	rs3212986
tr	AA 8 5 1.001 (0.275, 3.634) 0.999 7 5 6.825 (1.722, 27.044) 0.006
td	AA
td	8
td	5
td	1.001 (0.275, 3.634)
td	0.999
td	7
td	5
td	6.825 (1.722, 27.044)
td	0.006
tr	AC 17 18 0.590 (0.234, 1.489) 0.264 19 37 2.503 (0.977, 6.412) 0.056
td	AC
td	17
td	18
td	0.590 (0.234, 1.489)
td	0.264
td	19
td	37
td	2.503 (0.977, 6.412)
td	0.056
tr	CC 24 15 Ref. 8 39 Ref.
td	CC
td	24
td	15
td	Ref.
td	8
td	39
td	Ref.
table-wrap-foot	Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio.
footnote	Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio.
p	Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio.
sec	Genetic variation of ERCC1 single-nucleotide polymorphisms and sensitivity to cisplatin and carboplatin The frequencies of the genotypes of ERCC1-related SNPs (rs11615 and rs3212986) based on sensitivity to cisplatin and carboplatin are presented in Table 5. No correlation with the genotypes was found in these SNPs and sensitivity to cisplatin and carboplatin (P = 0.520 and P = 0.458, respectively) in malignant patients. Table 5 Frequency of genotypes at SNPs rs11615 and rs3212986 in the ERCC1 gene in malignant patients based on sensitivity to cisplatin and carboplatin Total Frequency Responders Non-responders OR (95% CI) P value PR+CR SD+PD Genotype (rs11615) Cisplatin TT 2 1 1 1.001 (0.041, 24.547) 0.999 TC 19 6 13 2.166 (0.334, 14.057) 0.418 CC 6 3 3 Ref. Carboplatin TT 3 2 1 0.375 (0.022, 6.348) 0.497 TC 3 1 2 1.500 (0.089, 25.392) 0.779 CC 7 3 4 Ref. Genotype (rs3212986) Cisplatin AA 5 3 2 0.667 (0.069, 6.409) 0.725 AC 14 3 11 3.667 (0.5557, 24.132) 0.177 CC 8 4 4 Ref. Carboplatin AA 4 1 3 3.000 (0.188, 47.963) 0.437 AC 3 2 1 0.500 (0.028, 8.952) 0.638 CC 6 3 3 Ref. Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio. CR: Complete response; PR: Partial response; SD: Stable disease; PD: Progressive disease.
title	Genetic variation of ERCC1 single-nucleotide polymorphisms and sensitivity to cisplatin and carboplatin
p	The frequencies of the genotypes of ERCC1-related SNPs (rs11615 and rs3212986) based on sensitivity to cisplatin and carboplatin are presented in Table 5. No correlation with the genotypes was found in these SNPs and sensitivity to cisplatin and carboplatin (P = 0.520 and P = 0.458, respectively) in malignant patients.
table-wrap	Table 5 Frequency of genotypes at SNPs rs11615 and rs3212986 in the ERCC1 gene in malignant patients based on sensitivity to cisplatin and carboplatin Total Frequency Responders Non-responders OR (95% CI) P value PR+CR SD+PD Genotype (rs11615) Cisplatin TT 2 1 1 1.001 (0.041, 24.547) 0.999 TC 19 6 13 2.166 (0.334, 14.057) 0.418 CC 6 3 3 Ref. Carboplatin TT 3 2 1 0.375 (0.022, 6.348) 0.497 TC 3 1 2 1.500 (0.089, 25.392) 0.779 CC 7 3 4 Ref. Genotype (rs3212986) Cisplatin AA 5 3 2 0.667 (0.069, 6.409) 0.725 AC 14 3 11 3.667 (0.5557, 24.132) 0.177 CC 8 4 4 Ref. Carboplatin AA 4 1 3 3.000 (0.188, 47.963) 0.437 AC 3 2 1 0.500 (0.028, 8.952) 0.638 CC 6 3 3 Ref. Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio. CR: Complete response; PR: Partial response; SD: Stable disease; PD: Progressive disease.
label	Table 5
caption	Frequency of genotypes at SNPs rs11615 and rs3212986 in the ERCC1 gene in malignant patients based on sensitivity to cisplatin and carboplatin
title	Frequency of genotypes at SNPs rs11615 and rs3212986 in the ERCC1 gene in malignant patients based on sensitivity to cisplatin and carboplatin
table	Total Frequency Responders Non-responders OR (95% CI) P value PR+CR SD+PD Genotype (rs11615) Cisplatin TT 2 1 1 1.001 (0.041, 24.547) 0.999 TC 19 6 13 2.166 (0.334, 14.057) 0.418 CC 6 3 3 Ref. Carboplatin TT 3 2 1 0.375 (0.022, 6.348) 0.497 TC 3 1 2 1.500 (0.089, 25.392) 0.779 CC 7 3 4 Ref. Genotype (rs3212986) Cisplatin AA 5 3 2 0.667 (0.069, 6.409) 0.725 AC 14 3 11 3.667 (0.5557, 24.132) 0.177 CC 8 4 4 Ref. Carboplatin AA 4 1 3 3.000 (0.188, 47.963) 0.437 AC 3 2 1 0.500 (0.028, 8.952) 0.638 CC 6 3 3 Ref.
tr	Total Frequency Responders Non-responders OR (95% CI) P value
td	Total Frequency
td	Responders
td	Non-responders
td	OR (95% CI)
td	P value
tr	PR+CR SD+PD
td	PR+CR
td	SD+PD
tr	Genotype (rs11615)
td	Genotype (rs11615)
tr	Cisplatin
td	Cisplatin
tr	TT 2 1 1 1.001 (0.041, 24.547) 0.999
td	TT
td	2
td	1
td	1
td	1.001 (0.041, 24.547)
td	0.999
tr	TC 19 6 13 2.166 (0.334, 14.057) 0.418
td	TC
td	19
td	6
td	13
td	2.166 (0.334, 14.057)
td	0.418
tr	CC 6 3 3 Ref.
td	CC
td	6
td	3
td	3
td	Ref.
tr	Carboplatin
td	Carboplatin
tr	TT 3 2 1 0.375 (0.022, 6.348) 0.497
td	TT
td	3
td	2
td	1
td	0.375 (0.022, 6.348)
td	0.497
tr	TC 3 1 2 1.500 (0.089, 25.392) 0.779
td	TC
td	3
td	1
td	2
td	1.500 (0.089, 25.392)
td	0.779
tr	CC 7 3 4 Ref.
td	CC
td	7
td	3
td	4
td	Ref.
tr	Genotype (rs3212986)
td	Genotype (rs3212986)
tr	Cisplatin
td	Cisplatin
tr	AA 5 3 2 0.667 (0.069, 6.409) 0.725
td	AA
td	5
td	3
td	2
td	0.667 (0.069, 6.409)
td	0.725
tr	AC 14 3 11 3.667 (0.5557, 24.132) 0.177
td	AC
td	14
td	3
td	11
td	3.667 (0.5557, 24.132)
td	0.177
tr	CC 8 4 4 Ref.
td	CC
td	8
td	4
td	4
td	Ref.
tr	Carboplatin
td	Carboplatin
tr	AA 4 1 3 3.000 (0.188, 47.963) 0.437
td	AA
td	4
td	1
td	3
td	3.000 (0.188, 47.963)
td	0.437
tr	AC 3 2 1 0.500 (0.028, 8.952) 0.638
td	AC
td	3
td	2
td	1
td	0.500 (0.028, 8.952)
td	0.638
tr	CC 6 3 3 Ref.
td	CC
td	6
td	3
td	3
td	Ref.
table-wrap-foot	Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio. CR: Complete response; PR: Partial response; SD: Stable disease; PD: Progressive disease.
footnote	Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio. CR: Complete response; PR: Partial response; SD: Stable disease; PD: Progressive disease.
p	Note. SNP: single-nucleotide polymorphism; CI: Confidence interval; OR: Odds ratio. CR: Complete response; PR: Partial response; SD: Stable disease; PD: Progressive disease.
sec	Discussion ERCC1 plays a fundamental role in DNA repair, and over 100 polymorphisms have been identified in the ERCC1 gene that can influence the structure and function of the encoded protein.19 In this case-control study, the rs11615-related TT genotype and the s3212986-related AA genotype of the ERCC1 gene were associated with a higher risk of lung cancer development in Iranian patients, with advanced NSCLC cases who have been treated with platinum-based chemotherapy. Furthermore, the relationship between the rs11615 SNP genotype and lung cancer risk was analyzed based on smoking status, revealing an association between lung cancer risk and AA and AC genotypes in non-smokers. Previous studies reported a correlation between ERCC1 polymorphisms and NSCLC susceptibility, which supports our results. For instance, Yu et al showed that NSCLC patients with the rs3212986 AA genotype in the ERCC1 gene have an increased risk of lung cancer, particularly among smokers.20 Gao also demonstrated that ERCC1 genotype rs11615 TT and genotype rs3212986 AA contribute to increased mortality risk from NSCLC. Additionally, a study involving Chinese patients indicated that the presence of the AA genotype at ERCC1 rs3213986 was associated with an increased risk of lung cancer, particularly in smokers.21 Other studies have suggested the association between various ERCC1 genotypes and the risk of lung cancer. Chaszczewska-Markowska et al confirmed the role of the rs11615 T allele and the rs3212986 GG genotype in NSCLC susceptibility.1 Likewise, two additional studies highlighted the predictive significance of the ERCC1 rs3212986 polymorphism in NSCLC patients.19,22 There is also evidence that certain SNPs within the ERCC1 gene can lead to decreased levels of ERCC1 mRNA and protein, which potentially impair cellular repair ability and ultimately promote cancer development.23-26 Moreover, several studies have confirmed a relationship between ERCC1 gene polymorphisms and NSCLC treatment efficacy.10,11,15,27,28 The results of a studyin Chinese patients revealed that individuals with the rs11615 TT genotype and the T allele or the rs3212986 AA genotype and the A allele had significantly lower response rates to treatment. As a result, these SNPs could serve as predictors of treatment response and may be associated with increased mortality in patients with advanced NSCLC.21 It has been suggested that rs11615 (ERCC1) and rs3738948 (ERCC3) can influence the efficacy of platinum-based chemotherapy in NSCLC patients.29 On the other hand, some studies have failed to find a significant relationship between ERCC1 gene polymorphisms and treatment efficacy in NSCLC patients receiving cisplatin-based chemotherapy. The results of a study among Iranian patients with colorectal and gastric cancers showed no significant association between genetic variations in the ERCC1 rs11615 SNP and response to oxaliplatin-based chemotherapy.30 Consistent with these findings, no significant effect of ERCC1 polymorphisms on treatment response was observed in NSCLC patients receiving cisplatin-based chemotherapy. These contradictory results may be due to differences in population, case selection, and study design. In our study, the relatively small number of NSCLC patients with homozygous genotypes may affect the power of statistical analysis to determine an association between ERCC1 polymorphisms and treatment response. Several limitations of the current study require consideration. First, the relatively small sample size, especially of lung cancer cases, restricted the statistical power for subgroup analysis; thus, the results should be confirmed with studies of larger populations. Second, this study only evaluated the risk of lung cancer, but investigating prognostic potential, especially by measuring survival parameters, could be valuable. Finally, further experiments are needed to elucidate the specific contribution of ERCC1 polymorphisms to the susceptibility to lung cancer susceptibility as well as to reveal other functional SNPs involved in DNA repair pathways. Highlights ERCC1 rs11615 and rs3212986 polymorphisms are associated with a higher risk of lung cancer. SNPs rs11615 and rs3212986 may serve as useful predictive markers to identify individuals at increased risk of lung cancer. The TT genotype of the rs11615 SNP was associated with a higher risk of NSCLC development.
title	Discussion
p	ERCC1 plays a fundamental role in DNA repair, and over 100 polymorphisms have been identified in the ERCC1 gene that can influence the structure and function of the encoded protein.19 In this case-control study, the rs11615-related TT genotype and the s3212986-related AA genotype of the ERCC1 gene were associated with a higher risk of lung cancer development in Iranian patients, with advanced NSCLC cases who have been treated with platinum-based chemotherapy. Furthermore, the relationship between the rs11615 SNP genotype and lung cancer risk was analyzed based on smoking status, revealing an association between lung cancer risk and AA and AC genotypes in non-smokers. Previous studies reported a correlation between ERCC1 polymorphisms and NSCLC susceptibility, which supports our results. For instance, Yu et al showed that NSCLC patients with the rs3212986 AA genotype in the ERCC1 gene have an increased risk of lung cancer, particularly among smokers.20 Gao also demonstrated that ERCC1 genotype rs11615 TT and genotype rs3212986 AA contribute to increased mortality risk from NSCLC. Additionally, a study involving Chinese patients indicated that the presence of the AA genotype at ERCC1 rs3213986 was associated with an increased risk of lung cancer, particularly in smokers.21 Other studies have suggested the association between various ERCC1 genotypes and the risk of lung cancer. Chaszczewska-Markowska et al confirmed the role of the rs11615 T allele and the rs3212986 GG genotype in NSCLC susceptibility.1 Likewise, two additional studies highlighted the predictive significance of the ERCC1 rs3212986 polymorphism in NSCLC patients.19,22
p	There is also evidence that certain SNPs within the ERCC1 gene can lead to decreased levels of ERCC1 mRNA and protein, which potentially impair cellular repair ability and ultimately promote cancer development.23-26 Moreover, several studies have confirmed a relationship between ERCC1 gene polymorphisms and NSCLC treatment efficacy.10,11,15,27,28 The results of a studyin Chinese patients revealed that individuals with the rs11615 TT genotype and the T allele or the rs3212986 AA genotype and the A allele had significantly lower response rates to treatment. As a result, these SNPs could serve as predictors of treatment response and may be associated with increased mortality in patients with advanced NSCLC.21 It has been suggested that rs11615 (ERCC1) and rs3738948 (ERCC3) can influence the efficacy of platinum-based chemotherapy in NSCLC patients.29 On the other hand, some studies have failed to find a significant relationship between ERCC1 gene polymorphisms and treatment efficacy in NSCLC patients receiving cisplatin-based chemotherapy. The results of a study among Iranian patients with colorectal and gastric cancers showed no significant association between genetic variations in the ERCC1 rs11615 SNP and response to oxaliplatin-based chemotherapy.30 Consistent with these findings, no significant effect of ERCC1 polymorphisms on treatment response was observed in NSCLC patients receiving cisplatin-based chemotherapy. These contradictory results may be due to differences in population, case selection, and study design. In our study, the relatively small number of NSCLC patients with homozygous genotypes may affect the power of statistical analysis to determine an association between ERCC1 polymorphisms and treatment response. Several limitations of the current study require consideration. First, the relatively small sample size, especially of lung cancer cases, restricted the statistical power for subgroup analysis; thus, the results should be confirmed with studies of larger populations. Second, this study only evaluated the risk of lung cancer, but investigating prognostic potential, especially by measuring survival parameters, could be valuable. Finally, further experiments are needed to elucidate the specific contribution of ERCC1 polymorphisms to the susceptibility to lung cancer susceptibility as well as to reveal other functional SNPs involved in DNA repair pathways.
sec	Highlights ERCC1 rs11615 and rs3212986 polymorphisms are associated with a higher risk of lung cancer. SNPs rs11615 and rs3212986 may serve as useful predictive markers to identify individuals at increased risk of lung cancer. The TT genotype of the rs11615 SNP was associated with a higher risk of NSCLC development.
title	Highlights
p	ERCC1 rs11615 and rs3212986 polymorphisms are associated with a higher risk of lung cancer.
p	SNPs rs11615 and rs3212986 may serve as useful predictive markers to identify individuals at increased risk of lung cancer.
p	The TT genotype of the rs11615 SNP was associated with a higher risk of NSCLC development.
sec	Conclusion In summary, the findings revealed that the ERCC1 rs11615 and rs3212986 polymorphisms are associated with a higher risk of lung cancer. These SNPs may serve as useful predictive markers to identify individuals at increased risk of lung cancer.
title	Conclusion
p	In summary, the findings revealed that the ERCC1 rs11615 and rs3212986 polymorphisms are associated with a higher risk of lung cancer. These SNPs may serve as useful predictive markers to identify individuals at increased risk of lung cancer.
sec	Authors’ Contribution Conceptualization: Yaser Masoumi-Ardakani, Abdollah Jafarzaheh, Behjat Kalantari Khandani. Data curation: Ali Khalouei, Arezu Khosravi Mashizi, Mohammadreza Zangouey, Mohammad Mehdi Yaghoobi, Beydolah Shahouzehi. Formal analysis: Mohammadreza Zangouey, Ali Khalouei. Funding acquisition: Yaser Masoumi-Ardakani. Investigation: Ali Khalouei, Yaser Masoumi-Ardakani. Methodology: Ali Khalouei, Arezu Khosravi Mashizi, Mohammad Mehdi Yaghoobi, Beydolah Shahouzehi. Project administration: Yaser Masoumi-Ardakani. Resources: Farnaz Sedghy, Behjat Kalantari Khandani. Software: Ali Khalouei, Mohammadreza Zangouey. Supervision: Yaser Masoumi-Ardakani. Validation: Yaser Masoumi-Ardakani, Farnaz Sedghy, Arezu Khosravi Mashizi, Mohammad Mehdi Yaghoobi. Visualization: Ali Khalouei, Arezu Khosravi Mashizi. Writing–original draft: Yaser Masoumi-Ardakani, Abdollah Jafarzaheh, Behjat Kalantari Khandani, Farnaz Sedghy, Arezu Khosravi Mashizi, Mohammad Mehdi Yaghoobi,Mohammadreza Zangouey, Beydolah Shahouzehi. Writing–review & editing: Yaser Masoumi-Ardakani, Farnaz Sedghy.
title	Authors’ Contribution
p	Conceptualization: Yaser Masoumi-Ardakani, Abdollah Jafarzaheh, Behjat Kalantari Khandani.
p	Data curation: Ali Khalouei, Arezu Khosravi Mashizi, Mohammadreza Zangouey, Mohammad Mehdi Yaghoobi, Beydolah Shahouzehi.
p	Formal analysis: Mohammadreza Zangouey, Ali Khalouei.
p	Funding acquisition: Yaser Masoumi-Ardakani.
p	Investigation: Ali Khalouei, Yaser Masoumi-Ardakani.
p	Methodology: Ali Khalouei, Arezu Khosravi Mashizi, Mohammad Mehdi Yaghoobi, Beydolah Shahouzehi.
p	Project administration: Yaser Masoumi-Ardakani.
p	Resources: Farnaz Sedghy, Behjat Kalantari Khandani.
p	Software: Ali Khalouei, Mohammadreza Zangouey.
p	Supervision: Yaser Masoumi-Ardakani.
p	Validation: Yaser Masoumi-Ardakani, Farnaz Sedghy, Arezu Khosravi Mashizi, Mohammad Mehdi Yaghoobi.
p	Visualization: Ali Khalouei, Arezu Khosravi Mashizi.
p	Writing–original draft: Yaser Masoumi-Ardakani, Abdollah Jafarzaheh, Behjat Kalantari Khandani, Farnaz Sedghy, Arezu Khosravi Mashizi, Mohammad Mehdi Yaghoobi,Mohammadreza Zangouey, Beydolah Shahouzehi.
p	Writing–review & editing: Yaser Masoumi-Ardakani, Farnaz Sedghy.
sec	Competing Interests The authors declare that there is no conflict of interests.
title	Competing Interests
p	The authors declare that there is no conflict of interests.
sec	Ethical Approval The current study was approved by the Ethics Committee of Kerman University of Medical Sciences and Health Services (IR.KMU.REC.1399.072), and all procedures were in accordance with standards set by the Declaration of Helsinki (1964).
title	Ethical Approval
p	The current study was approved by the Ethics Committee of Kerman University of Medical Sciences and Health Services (IR.KMU.REC.1399.072), and all procedures were in accordance with standards set by the Declaration of Helsinki (1964).
sec	Funding This study was financially supported by the Kerman Student Research Committee (Grant number: 98000685).
title	Funding
p	This study was financially supported by the Kerman Student Research Committee (Grant number: 98000685).

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