ATM gene polymorphisms are associated with poor prognosis of non-small cell lung cancer receiving radiation therapy

We investigated the prognostic significance of ataxia telangiectasia mutated (ATM) single nucleotide polymorphisms (SNPs) in 720 Han Chinese non-small cell lung cancer (NSCLC) patients who underwent radiation or chemoradiation therapy. Kaplan-Meier survival curves showed that overall survival (OS) and disease-free survival (DFS) rates were significantly associated with two ATM SNPs, rs664143 and rs189037. Patients with the rs664143 GA or AA genotype had poorer DFS (hazard ratio (HR) = 1.40, 95% confidence interval (CI) = 1.05–1.86, P = 0.021) and OS (HR = 1.28, 95%CI = 1.12–1.78, P = 0.040) than those with the rs664143 GG phenotype. Patients with the rs189037 AG/GG genotypes had poorer prognoses than those with the rs189037 AA genotype (AG/GG vs. AA: DFS, HR = 1.44, 95%CI = 1.06–1.95, P=0.019; OS, HR = 1.16, 95%CI = 1.16–1.17–2.21, P=0.004). These results were confirmed by subgroup analysis based on clinical factors such as smoking, histology, tumor stage, treatment, and radiation dose, all of which were significantly associated with DFS and OS rates in NSCLC patients. These findings show that ATM rs664143 and rs189037 variants determine prognosis in NSCLC patients that have undergone radiation or chemoradiation therapies.


INTRODUCTION
Lung cancer is one of the leading causes of cancer mortality worldwide, with nearly 2.1 million new lung cancer cases and 1.8 million lung cancer deaths reported in 2018 [1]. Lung cancer incidence in the United States has improved significantly in men, but remains high in the women [2]. In China, the incidence of lung cancer has increased by 26.9% annually, and the numbers of lung cancer patients have doubled every 10 to 15 years in the last 50 years [3]. Moreover, in the last three decades, the mortality rate of lung cancer has increased by 465%, and replaced liver cancer as the most common malignant tumor in China [4]. The two main categories of lung cancer are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC); NSCLC accounts for nearly 87% of all lung cancer cases, about 75% of which are diagnosed in the advanced stages with poor prognosis [5]. The 5-year survival rate of lung cancer patients is low and ranges from 5% to 31% [6,7]. Radiotherapy is one of the main treatment modalities, especially for NSCLC patients that are not amenable for surgery [8][9][10]. The prognostic indicators in NSCLC patients include TNM stage [11], serum levels of tumor

ATM SNPs and survival outcomes of NSCLC patients
Univariate analyses showed that two ATM SNPs, rs664143 and rs189037, as shown in Supplementary  Figure 1, were significantly associated with DFS and OS, but, the other two ATM SNPs, rs664677 and rs373759, did not show any significant correlation with survival outcomes ( Table 2).

AGING
We also compared the association between the number of risk alleles (rs664143 and 189037) and survival outcomes, DFS and OS. The results showed that patients with the risk alleles were associated with worse DFS and OS compared to those without a risk allele ( Table 2). As shown in Table 3, three SNPs (rs664143, rs664677, and rs373759) showed a prior false-positive probability of 0.1.

Subgroup analysis of ATM SNPs and NSCLC prognosis
We performed subgroup analysis with clinical factors such as smoking, histology, tumor stage, treatment, and radiation dose that are significantly associated with DFS and OS. The subgroup analysis showed that rs664143 and rs189037 were significantly associated with DFS and OS in both IIIA and IIIB stages ( Table  4 and  Table 5). Moreover, patients with rs664143 and rs189037 SNPs that were smokers and received radiation therapy (RT) with a radiation dose > 65 Gy showed worse survival outcomes than patients that were non-smokers and received chemo-radiation therapy (CRT) with a radiation dose ≤65 Gy, respectively (Tables 4, 5). Furthermore, based on histology, the rs664143 and rs189037 gene polymorphisms were associated with DFS and OS among the patients with adenocarcinomas (ADC) as shown in Tables 4, 5.

DISCUSSION
In this study, we assessed the association between four ATM SNPs and the survival outcomes in NSCLC patients of Han Chinese origin who received radiation therapy alone or chemo-radiation therapy. We demonstrated that two SNPs, rs664143 and rs189037, were significantly associated with DFS and OS rates in NSCLC patients treated with radiation or chemoradiation therapy. The association was significant with a prior false-positive rate of 0.1. NSCLC patients with rs664143 G or rs189037A alleles were at increased risk of disease progression compared with the other SNP genotypes. These results were further confirmed by the subgroup analysis. These findings suggest that these two ATM variants are associated with the prognosis of NSCLC patients and determine the efficacy of radiation therapy.
Radiation therapy induces DNA damage and tumor cell death. Inactivating ATM gene mutations contribute to genomic instability by reducing the efficiency of DNA double-strand break repair in response to radiation. ATM protein also detects DNA damage or other abnormal DNA structures in response to radiations and   AGING initiates DNA damage repair response that may include cell cycle arrest or apoptosis [21]. ATM activates checkpoint kinase, which induces phosphorylation of CDC25 at Ser216 and inhibits the cell cycle progression by suppressing the activity of CDC25 and M-Cdk. ATM also regulates the activity of p53, a critical tumor suppressor protein in multiple ways. It mediates p53 phosphorylation through Chk2 kinase activation. Moreover, ATM phosphorylates Mdm2 proto-oncogene and prevents its binding to p53. Furthermore, activation of p53 induces the expression of p21, which blocks the cell cycle in the G1-S phase by inhibiting CDK activity [22,23]. Therefore, inactivating mutations in the ATM gene can disrupt these critical cellular mechanisms. ATM inhibition enhances the sensitivity of cancer cells to radiation therapy [24,25], whereas, phosphorylated ATM protein increases the radiation resistance of cancer cells and correlates with poor prognosis of cancer patients [26]. Considering the importance of ATM kinase in DNA damage repair, our data suggests that activating or inactivating ATM gene polymorphisms influence the efficacy of radiation therapy and the prognosis of NSCLC patients.
Previous studies have shown that ATM gene polymorphisms are associated with poor prognosis of patients with pancreatic cancer, acute myeloid leukemia, and colorectal cancer [27,28,29]. Su et al. performed a case-control study of 230 NSCLC patients and showed that ATM rs664143 was not associated with the treatment response of patients with advanced NSCLC [30]. However, they did not evaluate its relationship with the prognosis of NSCLC patients. Du et al. showed that ATM rs664143 A and rs664677 C alleles were associated with poor prognosis of 412 esophageal squamous cell carcinoma (ESCC) patients that received radiation or chemoradiation therapy [31].
In the present study, we demonstrate that NSCLC patients with the rs664143 A allele is associated with unfavorable prognosis after receiving radiation or chemoradiation therapy. One probable mechanism explaining these outcomes is inaccurate splicing when rs664143 is combined with an intronic splicing enhancer or repressor [32]. However, further investigation is required to ascertain the molecular mechanisms involved in the process. We also demonstrate that ATM rs189037 is associated with poor prognosis of NSCLC patients treated with radiation or chemoradiation therapy. This SNP is located in the promoter region of the ATM gene. A previous study showed that ATM rs189037 is associated with radiationinduced pneumonia in lung cancer patients [33], but, its   [34]. Furthermore, pancreatic cancer patients with the rs664677 TC genotype showed worse prognosis than those with the rs664677 TT genotype [28]. In advanced ESCC patients receiving radiation therapy, two ATM SNPs, rs664143, and rs664677, were associated with survival times [31]. In contrast, our study showed no correlation between rs664677 and prognosis of NSCLC patients that received radiation or chemoradiation therapy. The reasons for these contrasting findings are not clear and further studies are necessary to determine the differential function or role of rs664677 in different types of cancers.
One plausible explanation for our findings is that the two ATM SNPs (or haplotypes) modulate the function of the ATM protein. This determines the response of NSCLC cells to radiation and chemo-radiation therapy, which affects their survival and contributes to differential prognosis of patients with different ATM SNPs. ATM protein is a key regulator of cell cycle. Cell cycle is blocked under conditions of DNA damage, incomplete replication, or abnormal spindle formation. When normal cells are irradiated, DNA repair mechanisms are activated so that the DNA damage is rectified. Cell cycle inhibition of irradiated cells prevents the proliferation of malignant cells that contain genetic mutations. However, tumor cells are defective in cell cycle checkpoints and DNA repair mechanisms. This results in uncontrolled proliferation and differentiation of tumor cells with genetic mutations [35]. Mutations in the ATM gene alter the structure and function of the ATM protein in ataxia telangiectasia (A-T) patients. Therefore, A-T patient cells show aberrant cell cycle checkpoints and DNA damage repair, increased sensitivity to apoptosis, chromosomal instability, and radiation sensitivity. Moreover, the incidence of cancers is significantly higher in patients with heterozygous or homozygous ATM mutations compared to individuals with the wild-type ATM gene [36]. The rs189037 is located at the 5'UTR of ATM gene (NC_000011.10: g. 108354934), and rs664143 is located at the intron of ATM gene (NC_000011.10: g.108354934). Two SNPs are very close loci (131bp) existing in two intros. We used the web-based tool (Improbizer, https://users.soe.ucsc.edu/~kent/improbizer/ improbizer.html) to check the splicing effect of two sites, and the results suggested that two sited exist in protein-binding motifs have a potential as binding sites of intronic splicing enhancer, indicating a possibility that both sites may be related to splicing process to lead to inaccurate splicing. However, this hypothesis should be confirmed through further research.
Our study has several limitations. Firstly, our study includes NSCLC patients treated with radiation or chemoradiation, but does not investigate NSCLC patients that underwent surgery. Secondly, we adjusted the survival outcomes for a few common clinical parameters, but, more factors should be considered. Thirdly, we did not identify the mechanisms underlying the findings of our study. Finally, we did not perform haplotype analysis because only two significant SNPs were found.

AGING
In conclusion, our study demonstrates that ATM gene polymorphisms are significantly associated with disease progression and survival outcomes in NSCLC patients that have received radiation or chemoradiation therapy. These two ATM SNPs are potential prognostic biomarkers to predict survival outcomes of NSCLC patients that receive radiation or chemoradiation therapy.

Study population
This We obtained written informed consent from all study subjects.

Radiation therapy
All patients underwent computed tomography (CT) scans of 5 mm slice thickness in a supine position with hands crossed before placing the forehead and thermoplastic body model fixation. The scans were performed from the mastoid process to the bottom of the lung and the images were transmitted over the network to a three-dimensional (3-D) treatment planning system. The target area delineation was according to ICRU50 and ICRU62 report guidelines. The gross tumor volume (GTV) was defined as the tumor volume seen below the lung window. The GTV node (GTVnd) was defined as a metastatic lymph node seen below the mediastinal window. The characteristics of the GTVnd were: short diameter > LCM; multiple fusions; necrosis or envelope invasion; and confirmed by positron emission tomography (PET) or mediastinoscopy. For concurrent chemoradiotherapy or radiotherapy alone, the double-lung V20 was limited to < 28% and < 30%, respectively, and adjusted according to the patient's physical condition, age, complications, and basic lung-function parameters. The other tissue limits included a maximum dose of the spinal cord of < 45 Gy, a cardiac V40 of < 30%, and an esophageal V50 of < 40-50%. A physician confirmed the treatment plan developed by the radiologist. The plan was further verified by calibration on the CT simulation positioning machine. Finally, the treatment was performed using a Vanaii linear accelerator.

Clinical data and follow-up
We obtained clinical data from the medical records of the patients for parameters such as age at treatment, gender, smoking, histologic type, TNM stage, treatment method, radiation technique, and radiation dose. The survival data was collected by follow-up through the telephone or from the outpatient medical records. The patients underwent physical examinations, CT or PET/CT, laboratory tests, and lung function evaluations at 6 weeks after therapy. The follow-up interval was every 3 months for 2 years, and 6 months thereafter. The primary follow-up outcome was overall survival (OS). OS was defined as the time period from the start of treatment to the last follow-up or death. We also evaluated disease-free survival (DFS), which was defined as the time period from the start of treatment until the date of the first local recurrence or metastasis at the last follow-up. Patients without progression were censored at the last follow-up date.

Selection and genotyping of ATM SNPs
We first searched for the ATM gene SNPs in the dbSNP database (https://www.ncbi.nlm.nih.gov/snp/) using a minor allele frequency of > 5%. Next, we selected tagSNPs with r 2 > 0.8 among the Han Chinese population using the International HapMap project database and identified four SNPs (rs664143, rs664677, rs189037, and rs373759) with moderate linkage disequilibrium by analyzing the 1000GENOMES project database.
We obtained 5 mL of fasting venous blood in ethylenediaminetetraacetic acid (EDTA)-coated tubes and isolated the genomic DNA from peripheral blood leukocytes using the Biospin Whole Blood Genomic DNA Extraction Kit (Bioer Technology Co., Ltd., China) according to the manufacturer's instructions. The samples were stored at −20°C.
The SNP sequences were PCR amplified using primer sequences that were designed using Primer Premier 5.0, and synthesized by Sangon Biotech (Shanghai, China) as shown in Supplementary Table 1. The PCR protocol was: initial denaturation cycle at 95°C for 7 min; 35 cycles of denaturation at 95°C for 1 min, annealing at 56°C for 1 min, and extension at 72°C for 1 min; final extension cycle at 72°C for 10 min. The PCR products were separated by 2% agarose gel electrophoresis, purified by ExoSAP-IT (USB Corp., Cleveland, OH, USA), sequenced in an Applied Biosystems 3730xl automated sequencer (Applied Biosystems, Foster City, CA, USA), and analyzed using the Vector NTI software.

Statistical analysis
The continuous variables were converted into categorical variables based on the mean age or median dose. The median OS and DFS were determined by Kaplan-Meir survival curve analyses using the log-rank test. The relationship between clinical parameters, ATM SNPs and survival parameters, OS and DFS, was determined using univariate and multivariate cox proportional hazards regression models. The multivariate Cox proportional regression model was adjusted for age, gender, smoking, histology, stage, treatment, radiation technique, and dose and the corresponding hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated. Subgroup analyses were performed for significant variables such as smoking, tumor stage, treatment, and radiation dose. The falsepositive probability analysis was conducted by assuming that the HR for a risk allele was 1.5 and the HR for a protective allele was 1/1.5 times below the prior probability of 0.01 for each SNP. A significant association was defined as a false-positive value of < 0.20. Statistical significance was defined as a twosided P-value < 0.05. Statistical analysis was performed using the SPSS 23.0 software.