Differences in EGFR and KRAS mutation spectra in lung adenocarcinoma of never and heavy smokers

Epidermal growth factor receptor (EGFR) mutations are common in lung adenocarcinomas of never smokers, while KRAS mutations are more frequent among heavy smokers. Different clinicopathological and biological characteristics may, therefore, exist in lung adenocarcinoma according to smoking status. In the present study, a retrospective review was performed using 521 patients with surgically resected lung adenocarcinomas. The clinicopathological factors of age, gender, pathological tumor size, nodal status, lymphatic permeation and blood vessel invasion and the EGFR and KRAS mutation spectra were compared between never and heavy smokers. EGFR mutations were detected in 233 (45%) patients, while KRAS mutations were detected in 56 (11%) patients. EGFR-mutated adenocarcinomas had a higher prevalence of females in the never smokers compared with the heavy smokers (P<0.001). KRAS-mutated adenocarcinomas had a higher prevalence of females (P<0.001) and showed less frequent vascular invasion (P=0.018) in the never smokers compared with the heavy smokers. Minor EGFR mutations, excluding exon 21 L858R and exon 19 deletions, were more common in heavy smokers than never smokers (P=0.055). KRAS G to A transition was more common in never smokers, while KRAS G to T and G to C transversions were more common in heavy smokers (P=0.036). The clinicopathological characteristics and the spectra of the EGFR and KRAS mutations in lung adenocarcinoma were different between the never and heavy smokers. Further large-scale studies are required to evaluate the efficacy of molecular targeting agents with consideration to specific EGFR and KRAS mutations.


Introduction
In lung cancer, epidermal growth factor receptor (EGFR) and KRAS mutations occur frequently in adenocarcinoma, rarely in squamous cell carcinoma and hardly ever in small cell carcinoma (1)(2)(3). These two mutations are known to have different correlations with smoking exposure. Specifically, EGFR-mutated adenocarcinomas are more common in individuals who have never smoked (1,3), while KRAS-mutated adenocarcinomas occur more frequently among heavy smokers (4). Therefore, we hypothesized that there may be different biological characteristics and etiologies between these types of lung adenocarcinomas related to cumulative exposure to tobacco smoke.
The presence of an EGFR mutation is the most important predictor of the efficacy of EGFR tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib (5,6). Activating mutations in the TK domain of EGFR are limited to exons 18-21, but show marked structural diversity, including point mutations, deletions and insertions (1,3,5,6). The L858R in exon 21 and small deletions in exon 19 have been shown to account for 80-90% of all EGFR mutations and are often termed as classic activating mutations (3). Although an inverse correlation between the extent of smoking and the frequency of these mutations has been shown by a number of investigators (7,8), the correlation between smoking exposure and other minor mutations remains unclear.
KRAS-mutated lung adenocarcinomas are more predominant in Caucasians (~30%) than East Asians (~10%) (1). These mutations serve as a useful biomarker of resistance to EGFR-TKIs (9), occurring frequently at codon 12 and occasionally at codon 13, but rarely located at codon 61 (2). Several organ-specific KRAS mutation genotypes have been reported. The majority are G to T transversions in lung adenocarcinoma and G to A transitions in colorectal cancer (10). Although there have been few studies investigating the correlation between smoking exposure and KRAS mutation genotypes, Miller et al (11) reported that never smokers were significantly more likely than former or current smokers to have G to A transition mutations than G to T or G to C transversion mutations.
The purpose of the present study was to elucidate the differences in the clinicopathological characteristics of EGFR-and KRAS-mutated lung adenocarcinomas and their related mutation spectra between never smokers and heavy smokers.

Materials and methods
Patients. Between February 2009 and March 2012, 667 patients with primary lung cancer underwent pulmonary resection. Among these, 521 patients with adenocarcinoma were selected for retrospective review and examined for EGFR and KRAS mutations using surgically resected specimens. There were 277 (53%) never smokers (≤5 pack-years), 53 (10%) light smokers (5-20 pack-years) and 191 (37%) heavy smokers (>20 pack-years). Induction chemotherapy, radiotherapy and pre-operative treatment with EGFR-TKIs were not performed for any of the patients in this series.
This study was conducted on specimens stored in the tissue bank, with the approval of the Institutional Review Board (IRB) of Juntendo University School of Medicine (Tokyo, Japan). According to the tissue bank protocol, in order to collect specimens for studies gaining approval by the IRB in the future, written consent was obtained from patients prior to surgery for the collection and storage of specimens during surgery. The contents of this study were deemed ethically acceptable and the IRB approved the use of the specimens stored in the tissue bank without obtaining new informed consent.
Molecular analysis. Genomic DNA was extracted from 3-5-mm 3 cubes of frozen fresh lung cancer tissue samples from surgically resected specimens. The peptide nucleic acid-locked nucleic acid (PNA-LNA) polymerase chain reaction (PCR) clamp method (12) was used to identify EGFR mutations: G719A, G719C and G719A in exon 18; all deletion genotypes in exon 19; T790M in exon 20; and L858R and L861Q in exon 21. The PNA-mediated PCR clamping method (13) was used to identify KRAS mutations: All genotypes at codon 12 and 13. Molecular analyses for the EGFR and KRAS mutations were conducted at Mitsubishi Chemical Medience Corporation (Tokyo, Japan).
Statistical analysis. The clinicopathological factors of age, gender, pathological tumor size, nodal status, lymphatic permeation and blood vessel invasion were compared between the never and heavy smokers, along with the EGFR and KRAS mutation spectra. A χ 2 test, Fisher's exact test or t-test was used for the statistical analysis. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using the SPSS statistical software package (version 20.0; SPSS, Inc., Chicago, IL, USA).

EGFR and KRAS mutations in all adenocarcinoma patients.
EGFR and KRAS mutations were detected in 233 (45%) and 56 (11%) of the total 521 lung adenocarcinoma patients, respectively. These two mutations were mutually exclusive. The point mutation L858R in exon 21 and deletions in exon 19 were detected in 118 and 98 tumors, respectively, and together, they accounted for 93% of all EGFR alterations. The remaining minor EGFR mutations included exon 18 G719A in eight tumors, exon 18 G719S in three tumors, exon 18 G719C in three tumors, exon 21 L861Q in four tumors and exon 20 T790M in four tumors. Double mutations were identified in  Table I. EGFR-mutated adenocarcinomas were more common in female never smokers than KRAS mutations.
Differences in characteristics of EGFR-and KRAS-mutated lung adenocarcinomas between never and heavy smokers. EGFR mutations were detected in 155 (67%) never smokers, 25 (11%) light smokers and 53 (23%) heavy smokers (Table II). EGFR-mutated adenocarcinomas had a higher female prevalence in never smokers compared with that in heavy smokers (P<0.001). Minor EGFR mutations, which exclude exon 21 L858R and exon 19 deletions, were more frequent in heavy smokers than in never smokers. However, this difference was not statistically significant (P=0.055).
KRAS mutations were detected in 11 (20%) never smokers, 8 (14%) light smokers and 37 (66%) heavy smokers (Table III). KRAS-mutated adenocarcinomas had a higher prevalence in females (P<0.001) and showed less frequent vascular invasion (P=0.018) in never smokers compared with heavy smokers. G to A transition was more common in never smokers, while G to T and G to C transversions were more common in heavy smokers (P=0.036).
Gender differences in the characteristics of EGFR-and KRAS-mutated lung adenocarcinomas. There were no significant gender differences in age, pathological nodal status, lymphatic permeation, blood vessel invasion or mutation spectra for either EGFR-or KRAS-mutated lung adenocarcinomas (Tables IV and V). The mean tumor size of the EGFR-mutated adenocarcinomas was significantly larger in males compared with females (P=0.027; Table IV). However, the corresponding gender difference was not significant for KRAS-mutated adenocarcinoma (P=0.802; Table V).

Discussion
Tobacco smoking is the main cause of lung cancer worldwide. Despite this, ~25% of worldwide (14) and 30-40% of Asian lung cancer patients (15) have never smoked. The occurrence of EGFR and KRAS mutations has been reported to be associated with smoking status. EGFR mutations are more commonly identified in the adenocarcinomas of never smokers (1,3), while KRAS mutations are more frequent in heavy smokers (4). Although no carcinogens causing EGFR or KRAS mutations in never smokers have yet been identified, the causes of each are suspected to differ based on smoking status. The present study showed notable differences in the clinicopathological characteristics and mutation spectra of lung adenocarcinoma based on cumulative exposure to tobacco smoke. It is noteworthy that minor EGFR mutations, which exclude exon 21 L858R and exon 19 deletions, tended to be more common in heavy smokers than in never smokers. However, it remains unclear why the incidence of exon 21 L858R and exon 19 deletions is so much higher than the various other TK domain mutations of EGFR (1,3). There should be specific mutagens (other than those associated with smoking) associated with these classic activating EGFR mutations. By contrast, the minor mutations of EGFR may randomly occur due to carcinogens present in tobacco smoke.
The correlation between EGFR-TKI efficacy and minor mutations is not fully clear. An in vitro study showed that gefitinib had variable growth-suppressive effects for different EGFR mutant-expressing cells (16). In a previous study of 62 lung cancer patients with minor EGFR mutations, Wu et al (17) observed a favorable response to EGFR-TKIs in 28 patients with point mutations on G719 and L861 and a poor response in 34 patients with different mutations. De Pas et al (18) also observed different antitumor activities of EGFR-TKIs according to the specific EGFR mutation genotype in a study of 10 lung cancer patients with minor mutations. The efficacy of EGFR-TKI treatment in patients with minor mutations is diverse and depends on the specific EGFR mutation.
KRAS and TP53 mutations in lung cancer have been reported to occur more frequently in smokers than in never smokers (4,19). Fewer studies have investigated mutation spectrum variations for KRAS according to smoking status than TP53 mutations, yet similar correlations between the two have been reported (19)(20)(21). Specifically, G to T transversion is most common in lung cancers in smokers, while G to A transition is more frequent among never smokers. Riely et al (21) demonstrated that not only G to T, but also G to C transversion frequently occurs in smokers with KRAS-mutated lung adenocarcinomas. The results of the current study are in agreement with these previous observations. The association between KRAS mutation and the lack of benefit of anti-EGFR monoclonal antibodies, such as cetuximab and panitumumab, in metastatic colorectal cancer has been established (22). By contrast, for advanced non-small cell lung cancer, the benefit of cetuximab has been shown regardless of KRAS mutation status in a phase III trial of cisplatin and vinorelbine treatment, with or without cetuximab (23,24). Notably, the KRAS mutation spectrum in colorectal cancer is quite different from that in lung cancer of smokers; G to T transversion is most common in lung cancer, while G to A transition is most common in colorectal cancer (10). The KRAS mutation spectrum may therefore be a better determinant of anti-EGFR monoclonal antibody efficacy than simply the presence or absence of the KRAS mutation. G to A transition is frequent not only in colorectal cancer, but also in lung cancer of never smokers, indicating that the same mutagen may be associated with their carcinogenic processes.
Gender differences in lung cancer susceptibility have been previously reported. Several studies have provided evidence for a biological role of estrogen in lung carcinogenesis by direct promotion of cell proliferation (25,26). Environmental risk factors including indoor air pollution from cooking-oil fumes (27) and coal burning (28) have been reportedly associated with high lung cancer incidence among Chinese women. However, in the present study, no gender differences were observed in the characteristics of the EGFR-or KRAS-mutated lung adenocarcinomas other than tumor size, indicating that smoking status has a greater effect on the cause of lung adenocarcinoma than gender differences in Japanese patients.
In conclusion, in the present study, the clinicopathological characteristics and the spectra of EGFR and KRAS mutations in lung adenocarcinoma were different between never and heavy smokers. These findings indicate that the causes of these mutations in lung adenocarcinomas are different in never and heavy smokers. The efficacy of molecular targeting agents may differ based on the mutation spectra of the targeted genes. Therefore, in the future, the efficacy of these agents should be evaluated based not only on the presence of the corresponding gene mutation in an individual tumor, but also on the specific mutation spectrum of each gene. Further large-scale studies are recommended to further investigate and validate these spectra.

Acknowledgements
This study was supported in part by a Grant-in-Aid for the Cancer Research from the Ministry of Health, Labour and Welfare of Japan and the Smoking Research Foundation.