Differences among lesions with exon 19, exon 21 EGFR mutations and wild types in surgically resected non-small cell lung cancer

The clinical behavior of patients with advanced non-small cell lung cancer (NSCLC) differ between epidermal growth factor receptor (EGFR) exon 19 deletion (Ex19) and EGFR exon 21 L858R mutation (Ex21). This study aimed to evaluate whether these differences exist in surgically resected NSCLC. A total of 198 patients with surgically resected NSCLC harbouring Ex19 (n = 53), Ex21 (n = 51), and EGFR wild-type (Wt) (n = 94) were analyzed. The clinicopathological features, laboratory parameters, recurrent sites and disease-free survival (DFS) were compared according to mutational EGFR status. Ex21 occurred more frequently in female (p < 0.001), never-smokers (p < 0.001), adenocarcinoma (p < 0.001), low grade (p = 0.013) than Wt lesions. Ex19 occurred more frequently in female (p = 0.016), never-smokers (p = 0.008), adenocarcinoma (p < 0.001), low grade (p = 0.025) than Wt lesions. Ex 21 lesions (p = 0.026) had larger lepidic components than Wt lesions. Wt lesions had larger mucinous variant components than Ex21 lesions (p = 0.045) and Ex19 lesions (p = 0.015). Ex21 lesions were associated with lower pretreatment neutrophil: lymphocyte ratio (NLR) than Wt lesions (p = 0.017). The recurrent sites and DFS were similar among patients with Wt, Ex19 and Ex21.

In the last decades, with the progress of targeted therapies in non-small cell lung cancer (NSCLC), the treatment paradigm has been changed for patients with metastatic NSCLC. The epidermal growth factor receptor (EGFR) gene mutations status are with key determinant when using small molecule tyrosine kinase inhibitors (TKIs) for NSCLC patients. EGFR mutations are found in 30% to 50% of lung adenocarcinomas, with the most common mutations being deletion in exon 19 (Ex19 in 45% patients) and a mutation in exon 21 L858R point (Ex21 in 40% patients). Both mutations are referred to as sensitizing EGFR mutations 1,2 . Based on the results of eight classic phase 3 randomized trials (IPASS, FIRST-SIGNAL, OPTIMAL, EURTAC, WJTOG3405, NEJ002, LUX-Lung 3, LUX-Lung 6) 3-10 in which both the first-generation (gifitinib, erlotinib) and second-generation (afatinib) of EGFR TKIs have demonstrated increased PFS and response rates than chemotherapy for patients harboring sensitive EGFR mutations, the role of EGFR TKIs have been established as first-line therapy for advanced NSCLC with sensitizing EGFR mutations.
Although the predictive effects of sensitive EGFR mutations-Ex19 and Ex21-are well defined, accumulating data have shown clinical differences between Ex19 and Ex21. Several studies have reported that patients with Ex19 had better survival outcomes than those with Ex21 in advanced NSCLC 11,12 . However, little reports has evaluated these differences in surgically resected NSCLC.
Correlation between EGFR mutation status and laboratory parameters. As shown in Table 2, laboratory parameters were compared among patients with the 3 EGFR statuses. Ex21 lesions were associated with lower pretreatment NLR than Wt lesions (p = 0.017). While there were no significant differences in CEA, HGB, PLT, or white cell among the 3 groups. adrenocortical metastasis, 2 patients had chest wall metastasis, and 2 patients had liver metastasis. There were no significant differences in recurrent or metastatic sites among the 3 groups (Table 3). Till December 31, 2015, 191 patients' DFS data were obtained and 7 patients' data were censored. The median follow-up was 30.0 months (ranging from 1.7 to 43.9 months). The median DFS were 33.6, 29.4, and 25.7 months for patients with Wt, Ex19, and Ex21 lesions, respectively. There were no significant difference in DFS among the three groups (p = 0.941) (Fig. 1).

Discussion
At present, we are entering the age of precise medicine for cancer treatment. In recent years, NSCLC, especially lung adenocarcinoma has been found to harbor mutations or rearrangements of specific driver oncogenes, which are used to predict the therapeutic effect of relevant targeted inhibitors 17,18 . The most landmark example is that the EGFR mutation status can predict the efficacy of EGFR TKIs. EGFR is a transmembrane receptor tyrosine kinase with extracellular ligand-binding domain, a lipophilic transmembrane region and an intracellular regulatory domain with tyrosine kinase activity. It has been demonstrated that the signaling pathways of EGFR are essential for different cell functions. Mutations of the EGFR genes may result in persistent activation of the tyrosine kinase which could promote proliferation, angiogenesis, invasion, and metastasis of tumor cells in NSCLC 19,20 . The two  most common sensitive mutations that account for more than 85% of all EGFR gene mutations are Ex19 and Ex21. Several researches have explored clinicopathological differences and prognostic value between Wt and Mt tumors [21][22][23][24] , till now, few studies have compared differences between Ex19 and Ex21. In this study, we for the first time investigate the differences of clinicopathological features as well as survival outcomes among Ex19, Ex21, and Wt lesions in surgically resected NSCLC.
Previous studies have demonstrated that EGFR mutations are commonly observed in a subset of NSCLC patients with the following features: nonsmoker, female, adenocarcinoma, and well-or moderately differentiated tumor cells 25,26 . In our study, Ex21 occurred more frequently in female, never-smokers, adenocarcinoma, and low grade tumors than Wt. While the frequencies of female, never-smokers, adenocarcinoma, and low grade tumors with Ex19 tumors were intermediate between the values for Ex21 and Wt tumors. In addition, Ex21 occurred more frequently in never-smokers than Ex19.
Neutrophils in tumor microenvironment have been shown to interact with tumor cells producing cytokines and chemokines, which influence tumor cell growth, angiogenesis and metastasis. In contrast to neutrophils,

Table 4. The association between adenocarcinoma histological subtypes and mutational statuses of EGFR in 161 patients with adenocarcinoma.
lymphocytes generally act as the host defense against tumor 27,28 . Elevated pretreatment NLR has been proved associated with the poor prognosis of patients with lung cancer 29 . For all we know, so far there has been none of study reported the correlation between NLR and EGFR mutation status. Our study for the first time demonstrated that Ex21 lesions were associated with lower pretreatment NLR than Wt lesions, while the proportion of lower NLR with Ex19 tumors were intermediate between the values for Ex21 and Wt tumors. It is currently believed that inflammatory cells in the tumor microenvironment play a significant role in tumor development. Further evaluation is necessary to examine the interaction and related mechanism between EGFR mutation status and host-derived stromal tissues as well as host immune cells.
The prognostic value of EGFR mutations in resected NSCLC remains controversial. Lee et al. 21 analyzed 117 patients with surgically resected pulmonary adenocarcinoma and found that patients with EGFR mutations had longer DFS than those with Wt. Similarly, D' Angelo et al. 30 analyzed 1118 patients with surgically resected NSCLC and found that patients with EGFR mutations had longer OS than those with Wt. Conversely, some studies revealed that EGFR mutation had no prognostic value for resected NSCLC. Liu et al. 23 investigated 131 patients with resected pulmonary adenocarcinoma and the result showed that there was no significant correlation between EGFR mutation status and DFS, OS. Several article reported different predictive and prognostic value between Ex21 and Ex19 in advanced NSCLC. Liu et al. 23 analyzed 131 patients with resected pulmonary adenocarcinoma and found that patients with Ex19 had longer DFS than those with Ex21. Conversely, Shigemastsu et al. 26 analyzed 62 patients with early-stage NSCLC who underwent resections and found that patients with Ex21 had longer survival time than those with Ex19. In our study, there were no significant difference in DFS among patients with Ex21, Ex19 and Wt. In addition, there were no significant differences in recurrent or metastatic sites among the 3 groups.
Several studies reported that Mt tumors comprised more commonly the InvAd-LP subtype of lung adenocarcinoma 14,16,32 . In our study, Mt tumors also more commonly comprised the InvAd-LP subtype than Wt tumors. Several studies reported that Mt tumors comprised more commonly the InvAd-MP subtype of lung adenocarcinoma 33,34 . In our study, Wt tumors more commonly comprised the InvAd-MV subtype. Few studies have compared histologic subtypes based on 2011IASLC/ATS/ERS classification between Ex19 and Ex21. Yoshizawa et al. 14 compared 48 Ex19 tumors and 36 Ex21 tumors and found that there were no significant differences in histologic subtypes of adenocarcinoma between Ex19 and Ex21. Villa et al. 35 reported that Ex21 tumors was associated with InvAd-LP subtype when they compared 22 Ex19 tumors and 12 Ex21 tumors. In our study, Ex21 mutations occurred at approximately twice the incidence rate of Ex19 mutations in InvAd-SP and InvAd-MV subtype tumors. Ex19 mutations occurred at approximately twice the incidence rate of Ex21 mutations in InvAd-PP subtype tumors. Both Ex19 and Ex21 lesions had smaller mucinous variant components than Wt lesions. Ex21 lesions had larger lepidic growth components than Wt lesions. While no significant difference in DFS was observed, the clinicopathological features were different among Wt, Ex19 and Ex21 in early-stage NSCLC who underwent resections. Both Ex19 and Ex21 occurred more frequently in female, never-smokers, adenocarcinoma, low-grade tumors than Wt lesions. Ex21 occurred more frequently in never-smokers than Ex19. Ex21 lesions were associated with lower pretreatment NLR than Wt lesions. Both Ex19 and Ex 21 lesions had smaller mucinous variant components than Wt lesions. Ex 21 lesions had larger lepidic growth components than Wt lesions. EGFR Mutation Analysis. Genomic DNA was isolated and purified from formalin-fixed paraffin-embedded tissues using the GTpure FFPE Tissue DNA Extraction Kit (GeneTech, Shanghai, China) in accordance with the manufacturer's instructions. A fragment method was used to detect Ex19/exon 20 insertion. Mt genes were amplified by polymerase chain reaction. To detect exon 18 mutations (G719X), and exon 21 mutations (L858R and L861Q), the Cycleave method was used based on the basic principle of realtime polymerase chain reaction. Each PCR assay contained forward and reverse primers (each 4 pmol), 2 μ l template DNA solution, and 2 units of Hot-Start Taq DNA polymerase (Takara, Shiga Japan) in a 40 ml volume. The PCR conditions consisted of initial Scientific RepoRts | 6:31636 | DOI: 10.1038/srep31636 denaturation at 95 °C for 3 min; 50 cycles of 95 °C for 15 s, annealing at 56 °C for 30 s and 72 °C for 30 s; and final extension at 72 °C for 5 min. The PCR products were sequenced using the Pyrosequencing PyroMark ID system (Qiagen, Hilden, Germany) following the manufacturer's instructions.

Clinicopathological Variables and Laboratory Parameters.
Clinicopathological data collected for analysis included age at diagnosis, gender, smoking history, tumor size, pathological TNM stage (the seventh edition of the lung cancer staging classification system), pathological types, tumor differentiation, pleural invasion, vessel invasion and histological subtypes of adenocarcinoma according to the 2011IASLC/ATS/ERS multidisciplinary classification of lung adenocarcinoma.
Statistical Analysis. Statistical analysis was performed using SPSS22.0 package. Continuous variables among the 3 EGFR mutation groups were compared using analysis of variance and post hoc comparisons test (Tukey test). We analyzed the association between categorical variables and EGFR mutation status using the Chi-Square test. Whenever it was possible and when the expected value in any of the tests was less than 5, the Fisher exact test was used. A 2-way analysis was performed in all comparisons. Survivals were analyzed using the Kaplan -Meier method and were compared using the log-rank test. Statistical significance was defined as when P < 0.05.