Histological subtype and smoking status, but not gender, are associated with epidermal growth factor receptor mutations in non-small‑cell lung cancer
- Authors:
- Published online on: December 23, 2013 https://doi.org/10.3892/mco.2013.232
- Pages: 252-258
Abstract
Introduction
Smoking, the major risk factor for lung cancer, is associated with all major histological subtypes, particularly small-cell and squamous cell lung carcinoma. By contrast, adenocarcinoma is the predominant subtype of lung cancer encountered in never-smokers (1–3). Global statistics estimated that 15% of lung cancer cases in males and 53% in females are not associated with smoking. Furthermore, among the East Asian populations, 11–23% of male and 61–90% of female lung cancer patients are never-smokers (1–7).
In lung cancer patients from Asia-Pacific countries, mutations in the epidermal growth factor receptor (EGFR) are the most common genetic aberrations (8–12) and are significantly associated with a good response to treatment with EGFR tyrosine-kinase inhibitors (TKIs). First-line EGFR-TKI therapy with gefitinib or erlotinib (13–17) in patients with lung adenocarcinoma harboring EGFR mutations may yield a response rate of ~70% and achieve a longer progression-free survival (PFS) (8–11,17). EGFR mutations are commonly associated with female gender, adenocarcinoma histology, never-smoker status and Asian ethnicity (9,13,14,18–21). Therefore, these characteristics have been integrated into clinical practice to guide treatment selection for patients with advanced lung cancer (13,14,22–24).
However, these EGFR mutation-associated clinicopathological characteristics are probably mutually interactive. For example, in Asian countries, the majority of female lung cancer patients are never-smokers and have tumors of the adenocarcinoma subtype, which makes it difficult to determine whether gender, smoking history or histological subtype is the decisive factor for EGFR mutations. The identification of the decisive factor associated with EGFR mutations is essential for designing preventive strategies and therapeutic interventions and for the elucidation of the processes underlying tumorigenesis in lung cancer. Therefore, it is critical to determine whether these well-established clinicopathological characteristics associated with EGFR mutations in NSCLC patients are confounded with each other. Thus, we retrospectively analyzed data retrieved from our institutes to elucidate the association between EGFR mutations and clinicopathological characteristics.
Patients and methods
Patients and variables
We retrospectively analyzed the clinicopathological characteristics of NSCLC patients who had adequate tumor tissue for EGFR mutation analyses of exons 18–21, between January, 2006 and August, 2011. Variables including age, gender, histological subtype, smoking history, disease stage and the presence and location of EGFR mutations were collected and analyzed. A patient was classified as a non-smoker if he/she had never smoked or had smoked <100 cigarettes during their lifetime. By contrast, an ever-smoker was defined as one who had smoked >100 cigarettes over their lifetime. For histological classification, we grouped the histology as adenocarcinoma and non-adenocarcinoma. Among the non-adenocarcinomas, 32 were squamous cell carcinomas, 28 were NSCLCs not otherwise specified, 4 were adenosquamous carcinomas and 1 was a large-cell carcinoma.
This study was approved by the Joint Institutional Review Board of Taipei Medical University (Taipei, Taiwan). Informed patient consent was obtained.
EGFR mutation analysis
EGFR mutations were determined using either direct sequencing or other previously described methods (25,26).
Statistical analysis
The association between EGFR mutation status and age, gender, histological subtype, smoking status and disease stage were evaluated using the Pearson’s χ2 test or the Fisher’s exact test when appropriate. Univariate (unadjusted) and multivariate logistic regression models (ULR and MLR, respectively) were used to delineate the effects of these established clinicopathological characteristics on EGFR mutations and the results were described as odds ratio (OR) with a 95% confidence interval (CI) and P-value. Furthermore, patients were stratified by smoking status to elucidate the association between EGFR mutations and the variables mentioned above. P<0.05 was considered to indicate a statistically significant difference. Data analyses were conducted using SPSS software version 15.0 (SPSS Inc., Chicago, IL, USA).
Results
Clinical characteristics and EGFR mutation status
We analyzed the clinical characteristics and EGFR mutation status of 426 NSCLC patients (Table I). Of the 426 patients, 359 (84%) had adenocarcinoma, 67 (16%) had non-adenocarcinoma NSCLC, 47% were females, 57% were non-smokers and 43% were ever-smokers. Of the 226 male patients, 73% were ever-smokers, whereas only 8% of the 200 female patients were ever-smokers. In total, 197 (47%) of the 426 patients were found to harbor EGFR mutations. Using the χ2 test, our results demonstrated that female gender, adenocarcinoma and non-smoking status were significantly associated with EGFR mutations in NSCLC (Table I). In detail, female patients had a higher EGFR mutation rate compared to males (62 vs. 33%, P<0.001) and non-smokers had a higher EGFR mutation rate compared to ever-smokers (63 vs. 25%, P<0.001). Patients with adenocarcinoma were more likely to harbor EGFR mutations compared to those with non-adenocarcinoma NSCLC (53 vs. 7%, P<0.001). In conclusion, our data were consistent with those reported by previous studies (9,13,14,18–21), demonstrating that gender, histological subtype and smoking are associated with EGFR mutations in a ULR analysis (Table II).
 | Table IILogistic regression analysis of clinicopathological characteristics as predictors of the presence of EGFR mutations. |
To eliminate the possible confounding effect, MLR analysis was used to evaluate the complex associations between these factors in the presence of EGFR mutations in NSCLC patients and demonstrated that gender was not an independent factor statistically associated with the presence of EGFR mutations in NSCLC patients (OR=1.25, P=0.416) (Table II). However, adenocarcinoma and never-smoker status were independently associated with the presence of EGFR mutations (OR=9.43, P<0.001 and OR=3.49, P<0.001, respectively).
EGFR mutation subtypes
In our study, 185 (93.9%) of the 197 EGFR mutations were either a deletion in exon 19 (del 19) (41.6%) or a single amino acid substitution in exon 21 (L858R, 52.3%). To elucidate whether the previously described clinicopathological factors were preferentially associated with specific EGFR subtypes, we performed logistic regression analyses in patients with L858R vs. those with del 19. We observed no preferential association of del 19 or L858R with any of the clinicopathological factors using either ULR or MLR analyses (Table III).
Patient stratification by smokimg status
To elucidate the complex effects of smoking and gender in the presence of EGFR mutations, we stratified all 426 NSCLC patients into non-smokers and ever-smokers (Table IV). Although 65% of the 182 non-smoking female patients were found to harbor EGFR mutations, which was a higher incidence compared to that observed among non-smoking male patients (56%), this difference was not statistically significant (P=0.204). Among ever-smokers, EGFR mutations were detected in 24% of male and 28% of female patients, although this difference was also not statistically significant (P=0.775). However, adenocarcinoma histological subtype was significantly associated with EGFR mutations in ever- and non-smokers (32 vs. 6%, P<0.001 and 66 vs. 13%, P<0.001, respectively) compared to non-adenocarcinoma NSCLC.
| Table IVAssociation of clinicopathological characteristics with the presence of EGFR mutations in patients classified by smoking status. |
To confirm that gender is a confounding factor associated with EGFR mutations, we analyzed the 359 patients with lung adenocarcinoma using an MLR model and determined that smoking, but not gender, was significantly associated with EGFR mutations in patients with lung adenocarcinoma (Tables V and VI).
| Table VAssociation of clinical characteristics with the presence of EGFR mutations in patients with adenocarcinoma. |
| Table VIAssociation of clinical characteristics with the presence of EGFR mutations in patients with lung adenocarcinoma stratified by smoking status. |
EGFR mutation testing
It was suggested that EGFR mutation testing is routinely performed in patients with lung adenocarcinoma in most countries (27–29). However, a number of NSCLC patients harboring EGFR mutations, who would potentially benefit from EGFR-TKI treatment (gefitinib or erlotinib) may be precluded if the decision to perform EGFR mutation testing is based solely on histological subtype, gender and smoking status. Our analysis demonstrated that 2.5–40.1% of NSCLC patients harboring EGFR mutations may not be identified if testing is selectively performed based upon these clinicopathological phenotypes (Table VII). In particular, if never-smoking status, female gender or never-smoking female patients with adenocarcinoma were used as the selection criteria for EGFR mutation testing, 22.8, 37.6 and 40.1% of NSCLC patients with EGFR mutations, respectively, would be precluded from EGFR-TKI treatment.
| Table VIIPatient populations tested and EGFR mutations precluded under different mutation testing strategies according to clinicopathological characteristics. |
Discussion
In this study, we investigated the different clinicopathological factors that are widely considered to be critically associated with EGFR mutations in NSCLC patients. We observed that, as opposed to the conclusions reported by previous studies, gender is not independently associated with the frequency of EGFR mutations in NSCLC patients (Tables II, V and VI) or with EGFR mutation subtypes (Table III). Furthermore, the current guidance of routine EGFR mutation analysis in NSCLC patients was validated.
Our conclusion seems to be discordant with the results of the majority of earlier studies (5,9,12,15). The univariate analysis with Pearson’s χ2 test or Fisher’s exact test, or the ULR model used in the present and the majority of previous studies (5,9,12,15), led to the conclusion that NSCLC patients characterized by female gender, never-smoking status and adenocarcinoma histology were more likely to harbor EGFR mutations (Table I). However, particularly in East Asian populations, the majority of female NSCLC patients have no history of smoking and their lung tumors are of the adenocarcinoma subtype, making the determination of the critical factors for EGFR mutations challenging. An MLR analysis may better elucidate the critical risk factors. In accordance with our results, supporting that gender is not associated with the frequency of EGFR mutations in NSCLC patients (Tables II and IV–VI), scattered pilot studies using MLR analyses to eliminate the confounding effects of these clinicopathological characteristics demonstrated that only adenocarcinoma and smoking history are significantly associated with EGFR mutations in NSCLC (30–32). Consistent with our findings (Tables V and VI), a recent study in the USA that enrolled 2,142 patients with lung adenocarcinoma revealed that the EGFR mutational frequency among female and male patients was not significantly different when patients were stratified into never- and ever-smokers (33). The results mentioned above collectively suggest that gender is not an independent factor associated with EGFR mutations in NSCLC patients.
Our findings may provide a rationale and clinical evidence to support the use of routine EGFR mutation testing prior to the administration of either EGFR-TKIs or chemotherapy as first-line treatment for lung adenocarcinoma, which is endorsed by the majority of available guidelines (27–29). Furthermore, our data demonstrated that 7% of non-adenocarcinoma NSCLC patients also harbor EGFR mutations. As shown in Table VII, up to 40.1% of NSCLC patients harboring EGFR mutations would be precluded from the significant benefit of EGFR-TKI treatment if mutational testing was restricted to female non-smokers with adenocarcinoma. Of equal importance, up to 32% of female non-smokers with adenocarcinoma (Table VII), who are commonly considered candidates for treatment with EGFR-TKIs, had no detectable EGFR mutations, indicating that first-line EGFR-TKI treatment would be harmful, rather than beneficial, to these patients. EGFR-TKI therapy in this population yielded meager response rates and a shorter PFS compared to those who received conventional platinum-based doublet chemotherapy (34), which may achieve a response rate of 23.5–31% (8,35).
Due to the significant differences in the percentage of EGFR mutations among East Asian and non-Asian NSCLC patients (~30 vs. 8%, respectively), ethnicity is considered to be significantly associated with EGFR mutation status (9,18). However, this observation may be confounded by clinicopathological parameters, such as histological subtype and smoking behavior. Our analysis (Table VIII) suggested that smoking status, but not ethnicity, is the major determinant for EGFR mutations in lung cancer among different populations. Additionally, the contribution from the cumulative smoking dose to the EGFR mutation frequency should also be considered. Recent studies demonstrated that the cumulative smoking dose is inversely correlated with the presence of EGFR mutations in lung adenocarcinoma (32,33), ranging from 4 to 34% in smokers with various numbers of smoking pack years (33). Therefore, the difference in the percentage of EGFR mutations among smokers (Table VI) may be attributed to the cumulative smoking dose.
| Table VIIIComparison of the presence of EGFR mutations in patients with adenocarcinoma stratified by smoking status. |
Another factor that may contribute to the differences in the incidence of EGFR mutations in never-smokers among different ethnic groups may be cumulative exposure to environmental tobacco smoke (ETS). Recently, household and workplace ETS, in particular, were reported to be inversely associated with the frequency of EGFR mutations in non-smokers with NSCLC (36). Specifically, the presence of EGFR mutations in lung adenocarcinoma was significantly different in never-smokers with and without ETS exposure (40.5 and 64.1%, respectively; P=0.015). Therefore, the difference in the percentage of EGFR mutations in either never- or ever-smokers among the two study groups (Table VIII) may correlate with the cumulative effect of direct or indirect exposure to smoking, suggesting that ethnicity may not be a crucial factor associated with EGFR mutations in NSCLC patients.
However, there were several limitations to this study. One limitation was the relatively small sample size, particularly the number of female smokers, which may affect our analyses. Since <10% of women are smokers and only 6.4% of female lung cancer patients are smokers in Taiwan (6), it is difficult to recruit an adequate number of female smokers in lung cancer studies. Furthermore, we were unable to present a comprehensive image on the EGFR mutation status of all the NSCLC patients, since not every patient had adequate tissue for mutation testing. Therefore, our findings require validation by further large-scale studies. The elucidation of the association between these factors and EGFR mutations may help identify factors that increase lung cancer susceptibility in the never-smoking population and design optimal preventive strategies.
In conclusion, this study demonstrated that gender is a confounding factor, whereas histological subtype and smoking status are independently and significantly associated with EGFR mutations in NSCLC patients. These findings support the current guidance that EGFR mutation testing should be routinely performed in patients with lung cancer of the adenocarcinoma subtype, regardless of their gender, and may indicate that gender may not be associated with tumorigenesis in NSCLC patients harboring EGFR mutations.
Acknowledgements
The authors would like to thank the Taipei Medical University and Hospital, the Center of Excellence for Clinical Trial and Research in Neuroscience, the Institute of Biomedical Sciences, Academia Sinica, National Yang-Ming University and the National Science Council, Executive Yuan, Taiwan, R.O.C. for their support.
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