Investigation of the epidermal growth factor receptor mutation rate in non-small cell lung cancer patients and the analysis of associated risk factors using logistic regression

The aim of the present study was to investigate the mutation rate of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) patients and to apply logistic regression analysis to investigate the factors associated with EGFR gene mutation to provide data for the treatment of NSCLC. Paraffin tissue, bronchoscopy or pleural effusion specimens were collected from 176 NSCLC patients following pathological diagnosis. The EGFR gene exon 19 delL747-S75linss and delL747-S752ins deletion mutations, and the exon 20 T790M and exon 21 L858R mutations were identified using amplification refractory mutation system analysis. The clinical data and laboratory results of the patients were collected, and the total mutation rate of the EGFR gene in exons 19, 20 and 21 in the 176 NSCLC patients was found to be 48.3% (85/176). In addition, the EGFR gene mutation rate in adenocarcinoma was found to be significantly higher than that in squamous cell and large cell carcinoma (χ2=12.454; P=0.002). Furthermore, the mutation rate was found to be significantly higher in females than in males (χ2=13.78; P=0.001). The rate of exon 19 mutation was 21.0% (37/176), whereas the rate of exon 20 T90M mutation was 1.7% (3/176) and that of exon 21 L858R mutation was 29.0% (51/176). The logistic regression analysis revealed that the female gender, adenocarcinoma, distant metastasis and chemotherapy are factors associated with EGFR gene mutation (P<0.05). The female gender resulted in an increased incidence (2.438 times that of males) of EGFR mutation. Similarly, adenocarcinoma, distant metastasis and chemotherapy exhibited an increase in EGFR mutation risk (by 2.571, 2.810 and 0.367 times, respectively). The rate of EGFR mutation was high in the NSCLC patients, predominantly in exons 21 and 19. Therefore, these factors (female gender, adenocarcinoma, distant metastasis and chemotherapy) may increase the probability of EGFR gene mutations.


Introduction
Lung cancer-related mortality is the highest among all the cancer types, and its incidence is gradually increasing (1). Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for 80% of all lung cancer cases, and includes squamous cell carcinoma, adenocarcinoma and large cell carcinoma. However, despite the continuous improvements in surgical resection, chemotherapy and radiation therapies, patients with lung cancer remain extremely vulnerable to relapse and mortality (2). The cure rate of lung cancer is extremely low and the average five-year survival rate of patients with lung cancer is <15% (3)(4)(5)(6).
At present, the treatment of cancer depends predominantly on cytotoxic chemotherapy, however, the efficacy in the majority of solid tumors remains extremely limited and marked toxic side-effects have been identified, particularly in patients with lung cancer (4)(5)(6). The early detection of lung cancer is not easy and patients are often diagnosed in the middle or late stages of lung cancer and thus, the opportunity for surgical treatment is missed and the conventional drugs for chemotherapy exhibit limited effects. However, gefitinib is a drug that exhibits improved efficacy and safety in locally advanced or metastatic NSCLC patients. Furthermore, the epidermal growth factor receptor (EGFR) gene mutation is a predictor of gefitinib sensitivity in lung cancer (7,8). Thus, early detection of the EGFR mutation rate and its associated factors in lung tumors may present an important reference to individualize clinical treatment and improve treatment implementation in lung cancer, as well as to reduce the toxicity  Cases, n  ---------------------------------------------------------------------------------------Project code (variable) EGFR mutation a EGFR non-mutation  (9)(10)(11). In the present study, the EGFR gene exon 19 deletion, the exon 20 T790M mutation and the exon 21 L858R mutation were detected, and single factor logistic regression analysis was used to further analyze the factors associated with the EGFR mutation, to provide data for the treatment of NSCLC.

Materials and methods
Subjects. Statistical analysis. A comparison between the two groups was performed by the Mann-Whitney U test or Student's t-test for measured variables. The differences between the rates of EGFR gene mutation were tested for statistical significance using χ 2 or Fisher's exact tests. The factors studied in patients with EGFR mutations were compared with the patients without EGFR mutations. Briefly, a univariate logistic regression analysis was conducted for each candidate variable and P<0.05 was considered to indicate a statistically significant difference. Next, a univariate logistic regression analysis was performed to identify which markers were associated with mutations of the EGFR gene (Table II). All data were analyzed using SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA). Univariate logistic regression analysis of factors associated with EGFR mutation. According to the clinical data, which included gender, age, histological type, clinical stage, degree of differentiation, lymph node metastasis, distant metastasis, pleural effusion, family history of cancer, occupational exposure and chemotherapy, a logistic regression was performed to identify the risk factors associated with EGFR mutation. The χ 2 test revealed that clinical stage (X3), degree of differentiation (X4), lymph node metastasis (X5), pleural effusion (X7), family history of cancer (X8), occupational exposure (X9), surgery (X11) and radiotherapy (X12) were not associated with an increased risk of EGFR mutation. However, gender (X1), pathological type (X2), distant metastasis (X6) and chemotherapy (X10) were identified as risk factors associated with EGFR mutation. In addition, a t-test revealed that age (X13) and tumor volume (X14) were not risk factors associated with EGFR mutation, whereas smoking index (X15) was identified as a risk factor (Tables I and III) The associated single factors were used to perform non-conditional logistic regression analysis, and the results revealed that Table III. T-test/Mann-Whitney U test results for EGFR mutation and EGFR non-mutation in NSCLC patients.    (Tables IV and V).

Discussion
Previous studies have shown that >70% of NSCLC patients with EGFR mutations are sensitive to EGFR-tyrosine-kinase inhibitor (TKI) drugs, compared with only 10% in patients without EGFR mutations (13). EGFR mutations occur predominantly in exons18-21 and it has also been observed that the EGFR-TKI drug class exhibits different clinical responses in patients with different EGFR mutation types. For example, an insertion mutation in exon 20 often exhibits resistance to gefitinib or erlotinib, whereas exon 18 mutations exhibit moderate sensitivity to these drugs (less than that of exon 19 or 21 mutations). Furthermore, the survival time of patients with exon 19 mutations is longer than that of patients with exon 21 mutations when receiving the same EGFR-TKI treatment (14)(15)(16). These findings indicate that it is important to adequately distinguish between EGFR mutation status and mutation types for the targeted therapy of NSCLC.
In addition, significant differences in the EGFR mutation rate in NSCLC patients have been identified between Asian and non-Asian populations with rates of 26-40% and 2-12%, respectively. Furthermore, almost all mutations detected in adenocarcinoma tissue (17)(18)(19)(20)(21), were in the tyrosine kinase domains of 19 and 21 of exon, which usually account for ~90% of EGFR mutations. These two sites of mutation were also found to significantly correlate with the efficacy of EGFR-TKI treatment. Therefore, the accurate detection of genetic changes plays a decisive role in the clinical treatment of the two sites.
The present study revealed that in 176 NSCLC patients, the total mutation rate of the EGFR gene exons 19, 20 and 21 was 48.3% (85/176). In addition, the adenocarcinoma EGFR gene mutation rate (77/139) was significantly higher than that in squamous cell (8/36) and large cell (0/1) carcinoma. The total EGFR gene mutation rate was marginally higher than that reported in previous studies, however, the majority of mutations were detected in adenocarcinoma, which is consistent with previously reported results (19). This study also revealed that the mutation rate in females (51/78) was significantly higher than that in males, consistent with the results reported by Toyooka et al (22). The reasons for this remain unclear, however, it may be associated with different lifestyles, smoking habits and endocrine factors.
Previous studies have shown that the factors associated with EGFR gene mutations in NSCLC patients are not entirely consistent, and a small number of studies have reported contradicting conclusions (9)(10)(11). In the current study, univariate analysis revealed that gender, pathological type, distant metastasis, chemotherapy and smoking index are factors associated with EGFR gene mutations. Furthermore, unconditional logistic regression analysis was used and revealed that female gender, adenocarcinoma, distant metastasis and chemotherapy are also factors associated with EGFR gene mutations. The results showed that females have an increased incidence (2.438 times that of males) of EGFR mutation. Similarly, adenocarcinoma, distant metastasis and chemotherapy were found to exhibit an increased risk of EGFR mutation by 2.571, 2.810 and 0.367 times, respectively.
In conclusion, the EGFR gene mutation rate is higher in patients with NSCLC, predominantly in exons 21 and 19. This study provides specifc data for the study of EGFR mutations in lung cancer research and treatment.