Figures
Abstract
Epidermal growth factor receptor (EGFR) mutations are the strongest response predictors to EGFR tyrosine kinase inhibitors (TKI) therapy, but knowledge of the EGFR mutation frequency on lung adenocarcinoma is still limited to retrospective studies. The PIONEER study (NCT01185314) is a prospective molecular epidemiology study in Asian patients with newly diagnosed advanced lung adenocarcinoma, aiming to prospectively analyze EGFR mutation status in IIIB/IV treatment-naïve lung adenocarcinomas in Asia. We report the mainland China subset results. Eligible patients (≥20 yrs old, IIIB/IV adenocarcinoma and treatment-naïve) were registered in 17 hospitals in mainland China. EGFR was tested for mutations by amplification refractory mutation system using biopsy samples. Demographic and clinical characteristics were collected for subgroup analyses. A total of 747 patients were registered. Successful EGFR mutation analysis was performed in 741, with an overall mutation rate of 50.2%. The EGFR active mutation rate is 48.0% (with 1.3% of combined active and resistance mutations). Tobacco use (>30 pack-year vs. 0–10 pack-year, OR 0.27, 95%CI: 0.17–0.42) and regional lymph nodes involvement (N3 vs. N0, OR 0.47, 95%CI: 0.29–0.76) were independent predictors of EGFR mutation in multivariate analysis. However, even in regular smokers, the EGFR mutation frequency was 35.3%. The EGFR mutation frequency was similar between diverse biopsy sites and techniques. The overall EGFR mutation frequency of the mainland China subset was 50.2%, independently associated with the intensity of tobacco use and regional lymph nodes involvement. The relatively high frequency of EGFR mutations in the mainland China subset suggest that any effort to obtain tissue sample for EGFR mutation testing should be encouraged.
Citation: Shi Y, Li J, Zhang S, Wang M, Yang S, Li N, et al. (2015) Molecular Epidemiology of EGFR Mutations in Asian Patients with Advanced Non-Small-Cell Lung Cancer of Adenocarcinoma Histology – Mainland China Subset Analysis of the PIONEER study. PLoS ONE 10(11): e0143515. https://doi.org/10.1371/journal.pone.0143515
Editor: Karl X. Chai, University of Central Florida, UNITED STATES
Received: April 16, 2015; Accepted: November 5, 2015; Published: November 23, 2015
Copyright: © 2015 Shi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This study was funded by AstraZeneca and partly supported by Chinese National Major Project for New Drug Innovation (2008ZX09312, 2012ZX09303012-001) and Research Special Fund for Public Welfare Industry of Health (200902002-1).
Competing interests: The authors have declared that no competing interests exist.
Introduction
Lung cancer is the leading cause of cancer-related death in the world [1]. Platinum-based chemotherapy remain the main treatment choice for advanced non-small-cell lung cancer (NSCLC) [2]. However, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) therapy recently achieved promising successes in NSCLC patients harboring EGFR active mutations [3–5], significantly prolonging patients’ survival [6,7].
Therefore, it is of great importance to determine the prevalence of EGFR mutations frequency. Studies in different populations identified some subgroups (adenocarcinoma histology, women, never-smokers and East Asian ethnic origin) with higher EGFR active mutation rate [8,9]. However, several retrospective studies in resectable and advanced lung adenocarcinoma have suggested that gender was not an independent factor for EGFR mutation [10,11]. Therefore, it is significant to identify demographic and clinical characteristics associated with EGFR mutations, to allow identifying subpopulations of patients at high risk of harboring EGFR mutation, thus allowing the oncologists to decide which first-line treatment may offer the most benefits.
Most studies assessed EGFR mutation status in a clinical trial setting, or retrospectively based on archival tissue samples which might not sufficiently comply with real-life practice on an epidemiological point of view. Therefore, prospective studies of EGFR mutation status in clinical settings are needed.
A prospective epidemiological study has been conducted in Spain to screen for EGFR mutations in lung cancer [12], but no prospective studies were carried out for Asian patients. The PIONEER study (NCT01185314) is a large prospective molecular epidemiology study in Asian patients with newly diagnosed advanced lung adenocarcinoma, aiming to assess their EGFR mutation status [13]. This study enrolled a total of 1510 patients in 51 investigational sites in 7 Asian countries/regions (mainland China, Hong Kong, India, Philippines, Taiwan, Thailand and Vietnam) to investigate EGFR mutation frequency and to correlate these mutations with patients’ demographic and clinical characteristics, as well as with various tissue sampling techniques. In the present report, we present the results for the subset of patients from mainland China.
Material and Methods
Study Design and Patients
The PIONEER study (NCT01185314) is an epidemiological, multicenter, open-label and non-comparative study of EGFR mutation status in patients with newly diagnosed advanced (stage IIIb/IV) NSCLC.
Eligibility criteria were: 1) ≥20 years old; 2) histologically or cytologically confirmed advanced lung adenocarcinoma (stage IIIb/IV); and 3) treatment-naïve. Patients were registered in 17 hospitals in mainland China.
The study was performed according to the Declaration of Helsinki and good clinical practice guidelines. The study was approved by the Ethics Committees of all study centers. All patients provided a written informed consent for participation in the study and the use of tumor samples (tissue or cytology).
Data collection
Demographic data of patients were collected, including gender, age, and smoking status. Disease data included date of first NSCLC diagnosis, histological type, AJCC stage, nodal status, and distant metastases. Standardized case report forms were used to record the data in accordance with the protocol’s instructions. Smoking was assessed using two ways. First, patients were classified according to their actual smoking status (never-smoked means that the subject smoked no cigarettes during his entire lifetime; ex-smoker means that the subject no longer smokes; occasional smoker means that the subject smokes, but not every day; and regular smoker means that the subject smokes every day). Second, smoking patients were classified according to their tobacco consumption, in pack-year.
EGFR Mutation Analysis
Tumor samples were obtained from primary or metastatic lesions, and were handled and stored following laboratories' quality control requirements. Biopsy site and technique were recorded. Cytological samples were accepted only when histological material was unavailable. After tumor DNA extraction, EGFR mutation was analyzed at Laboratory of Medical Oncology, Cancer Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College; Beijing Key Laboratory of Clinical Study on Anticancer Molecular Targeted Drugs by an amplification refractory mutation system (ARMS)-based EGFR mutation detection kit (EGFR RGQ PCR kit, Qiagen, Crawley, UK). This kit allows the detection of 29 mutations in the EGFR gene.
Statistical Analyses
Statistical analysis was performed by the Statistics and Programming department of Astra Zeneca Japan using SAS 8.2 (SAS Institute, Cary, NY, USA). The per protocol analysis (PPS) set was used for all statistical analyses. Mutations prevalence and corresponding 95% confidence intervals (95%CI) were calculated using the Wilson score method. Associations between mutations and demographic and clinical characteristics were analyzed by χ2 tests or Fisher’s exact tests, as appropriate. Characteristics associated with mutations with a P-value <0.05 were included in a multivariate logistic model. All analyses were two-sided and a P-value <0.05 was considered significant. In the multivariate analysis, a P-value <0.01 was considered significant.
Results
Baseline characteristics of the study population
From September 2010 to July 2011, a total of 747 patients (747/1510, 49.5% of the overall study population) from 17 hospitals in mainland China were registered for analysis (PPS). Among them, 397 (53.1%) were men and 350 (46.9%) were women. The mean age at registration was 57.4±11.4 years, ranging from 20 to 83 years. All patients were Chinese population. The total number of never-smokers was 421 (56.4%), ex-smokers were 136 (18.2%), occasional smokers were 19 (2.5%) and regular smoker were 171 (22.9%). Among patients with a positive smoking history (n = 324), the mean pack-years was 32.9±24.0. For 97.2% (n = 726) of patients, the time interval between original diagnosis and enrolment into the study was <6 months. At original diagnosis, distant metastases were found in 615 (82.3%) patients, and 132 (17.7%) patients suffered from a locally advanced disease. The most common site for biopsy was lung (549 patients), local lymph nodes (77 patients) and distant lymph nodes (47 patients). The most common method of biopsy was image-guided core biopsy (222/747, 29.7%), bronchoscopic biopsy (180/747, 24.1%) and incisional biopsy (95/747, 12.7%). In the majority of patients, tissue sample was taken from the primary tumor (n = 541, 72.4%) and was fixed in 10% neutral buffered formalin (n = 639, 85.5%).
Factors associated with EGFR mutation
In the PPS population, EGFR mutation analysis was successful in 741 patients (99.2%). Among the 741 evaluable patients, 372 (50.2%, 95%CI: 46.6–53.8) were EGFR mutation-positive (M+), and 369 (49.8%, 95%CI: 46.2–53.4) were EGFR mutation-negative (M-). In univariate analyses, as expected, the female gender was significantly associated with higher EGFR mutation frequency (60.6% for female vs. 41.0% for male, P<0.001), and smoking status was associated with M+, with a higher incidence of EGFR M+ in patients who never smoked (59.6%, P<0.001) (Fig 1) or those who smoked 0–10 pack-years (58.4%, P<0.001) (Table 1). As smoking and pack-years were covariates, only the pack-year variable was included in the multivariate analysis. Stage classification (53.0% for stage IV vs. 38.6% for stage IIIb, P = 0.002) and regional lymph node involvement (62.1% for N0, 52.6% for N1-2 and 43.8% for N3, P = 0.009) were also factors associated with EGFR mutation frequency in univariate analyses.
Age, time from original diagnosis, malignant pleural effusion, and primary tumor were not associated with M+ status. Tumor grade seemed to be significantly associated with EGFR mutation frequency; however, this variable was documented for a little number of patients, mainly due to the limited amount of tissue available for diagnosis, and was therefore not included in the multivariate analysis. The distribution of EGFR M+ according to biopsy site, type and technique was relatively similar (S1 Table). Patients with lymph nodes biopsy didn’t show lower EGFR mutation frequency compared with other biopsy sites (47.2% vs 50.8%, P = 0.4591).
Multivariate analysis identified pack-year and regional lymph node involvement as independent predictors of EGFR M+. Patients smoking 10–30 (OR 0.54, 95%CI: 0.37–0.78) or more than 30 (OR 0.27, 95%CI: 0.17–0.42) pack-year had less chance to harbor an EGFR mutation than those smoking less than 10 pack-year. Tumors with regional lymph nodes involvement (stage N1-2, OR 0.71, 95%CI: 0.44–1.14; and N3, OR 0.47, 95%CI: 0.29–0.76) were also less likely to harbor EGFR mutations than patients without lymph nodes involvement (N0) (Table 2).
Types of EGFR mutation
The most common mutations were exon 19 deletion and L858R point mutation, observed in 182 (24.6%) and 169 (22.8%) patients, respectively. Out of the 372 EGFR M+ patients, 346 patients (46.7%) had EGFR active mutations alone, 16 patients (2.2%) had resistant mutations alone, and 10 patients (1.3%) had both active and resistant EGFR mutations (Table 3). Pack-year and regional lymph nodes involvement were also identified as independent factors significantly associated with EGFR active mutations by univariate and multivariate analysis (S2 Table and S3 Table).
Additionally, nine patients provided both histology and cytology samples, among which one patient had mutation results that did not match.
Discussion
EGFR mutations are currently the strongest response predictor to EGFR-TKI therapy [14]. In addition, with the establishment of first-line treatment using EGFR-TKI [6,7], the knowledge of EGFR mutation status in treatment-naïve advanced NSCLC, especially lung adenocarcinoma, is of great importance for clinical decision. Previous data were generally derived from retrospective studies and clinical trials, which might not reflect real-life. PIONEER is a large epidemiological study prospectively investigating the EGFR mutation status in Asian treatment-naïve advanced NSCLC patients [13], and we report the results from the mainland China subset.
In our study (PPS population), the overall EGFR mutation frequency was 50.2%, similar to previous studies in East Asian patients [6,15], but much higher than in Caucasian lung adenocarcinoma patients [12,16]. Besides a different ethnicity distribution, EGFR mutations are also suggested to be more common in adenocarcinoma, women, and never-smokers [8,9]. Rekhtman et al. reported that “pure” squamous cell lung cancer, verified by immunohistochemistry as Np63 (+)/TTF-1(-), lacked EGFR mutations [17], thus leaving lung adenocarcinoma as the major candidate for EGFR screening in NSCLC. In the present prospective study, we also found that the impact of gender was not significant after adjustment for smoking status, and did not have any interaction with smoking (data not shown).
Smoking status was identified as an independent predictor of EGFR mutation in the present study. We observed that the EGFR mutation incidence decreased with the increasing tobacco consumption, which is similar to a retrospective by Li et al. in resectable NSCLC [18]. When smoking was classified into never-smoked, ex-smoker, occasional smoker, and regular smoker according to their smoking frequency, there was a wide gap between never-smoked and ever-smoked patients. However, even in regular smokers, EGFR mutation was still found in 35.3% patients, indicating the necessity of EGFR mutation testing in this group of patients.
Another interesting finding of the present study was that EGFR mutation frequency independently decreased with the involvement of regional lymph node, and patients with N3 disease had the least chance of harboring an EGFR mutation. This finding is supported by a study by Na et al. in a Korean population (OR 0.2, 95%CI: 0.0–0.7, P = 0.02) [19]. There is little chance that this observation might be due to the tumor sample volume because the ARMS approach has a well-known detection sensitivity, and the EGFR M+ frequency according to biopsy sites was comparable. The plausible explanation still warrants further investigations.
EGFR T790M mutation is associated with shorter progression-free survival with EGFR-TKI therapy in patients with NSCLC [20,21]. However, the prevalence of the EGFR T790M mutation varied by population and technique. Su et al. detected T790M in 2.8% of Chinese patients with TKI-naïve NSCLCs [20], while Rosell et al., using a Taqman assay, reported that 35% of EGFR M+ (L858R or 19 del) NSCLC had concomitant T790M mutation in Caucasians [21]. In this prospective cohort of treatment-naïve advanced lung adenocarcinoma, the T790M mutation was found in only 0.4% of our Chinese patients using the ARMS assay, accounting for 0.8% of EGFR M+ patients, all of which were observed concomitantly with the EGFR L858R mutation. This rate of the T790M mutation is much lower than the frequency observed by Su et al. (2.8%) [20]. This might be due to the smaller sample size in their study, and by the different detection method. The EGFR exon 20 insertion mutation was also suggested to induce a poor responses to EGFR-TKI [22], and was reported in about 2% of NSCLC or lung adenocarcinoma [23–25], which is similar to our report (2.0%).
The present study has some limitations. Indeed, despite all our efforts, we were unable to recruit all lung cancer patients, mainly because some of them did not consent, which could introduce a bias. Furthermore, tobacco consumption was self-reported, and might suffer from some under-reporting.
Conclusion
In conclusion, from the PIONEER prospective study, the overall EGFR mutation frequency of the mainland China subset was 50.2%. The EGFR active mutation rate is 48.0% (with 1.3% of combined active and resistance mutations). The relatively high frequency of EGFR mutation in this population and the comparable EGFR M+ rate among diverse biopsy sites and techniques suggest that any effort to gain tissue samples for EGFR mutation testing should be encouraged.
Supporting Information
S1 Table. Overall analysis of EGFR mutation test by sample type (PPS).
https://doi.org/10.1371/journal.pone.0143515.s001
(DOC)
S2 Table. Subgroup analysis for EGFR active mutations alone (PPS).
https://doi.org/10.1371/journal.pone.0143515.s002
(DOC)
S3 Table. Logistic model analysis for EGFR active mutations alone (PPS).
https://doi.org/10.1371/journal.pone.0143515.s003
(DOC)
Acknowledgments
The authors thank the patients and investigators for their participation in this study. The authors thank the PIONEER study PI, Pan-Chyr Yang for the permission to separately report the mainland Chinese results.
Author Contributions
Conceived and designed the experiments: YKS. Performed the experiments: YKS JLL SCZ MZW SJY NL GW WL GQL KCC LAC MZZ PY XWW YPL QSG LGN JWL XHH. Analyzed the data: YKS JLL SCZ MZW SJY NL GW WL GQL KCC LAC MZZ PY XWW YPL QSG LGN JWL XHH. Contributed reagents/materials/analysis tools: YKS JLL SCZ MZW SJY NL GW WL GQL KCC LAC MZZ PY XWW YPL QSG LGN JWL XHH. Wrote the paper: YKS JLL SCZ MZW SJY NL GW WL GQL KCC LAC MZZ PY XWW YPL QSG LGN JWL XHH.
References
- 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61: 69–90. pmid:21296855
- 2. Goldstraw P, Ball D, Jett JR, Le Chevalier T, Lim E, Nicholson AG, et al. Non-small-cell lung cancer. Lancet. 2011;378: 1727–1740. pmid:21565398
- 3. Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004;350: 2129–2139. pmid:15118073
- 4. Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 2004;304: 1497–1500. pmid:15118125
- 5. Pao W, Miller V, Zakowski M, Doherty J, Politi K, Sarkaria I, et al. EGF receptor gene mutations are common in lung cancers from "never smokers" and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A. 2004;101: 13306–13311. pmid:15329413
- 6. Mok TS, Wu Y-L, Thongprasert S, Yang C-H, Chu D-T, Saijo N, et al. Gefitinib or carboplatin–paclitaxel in pulmonary adenocarcinoma. New England Journal of Medicine. 2009;361: 947–957. pmid:19692680
- 7. Zhou C, Wu YL, Chen G, Feng J, Liu XQ, Wang C, et al. Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study. Lancet Oncol. 2011;12: 735–742. pmid:21783417
- 8. Mitsudomi T, Yatabe Y. Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer. Cancer Sci. 2007;98: 1817–1824. pmid:17888036
- 9. Zhang Y, Sun Y, Pan Y, Li C, Shen L, Li Y, et al. Frequency of driver mutations in lung adenocarcinoma from female never-smokers varies with histologic subtypes and age at diagnosis. Clin Cancer Res. 2012;18: 1947–1953. pmid:22317764
- 10. Wu SG, Gow CH, Yu CJ, Chang YL, Yang CH, Hsu YC, et al. Frequent epidermal growth factor receptor gene mutations in malignant pleural effusion of lung adenocarcinoma. Eur Respir J. 2008;32: 924–930. pmid:18508816
- 11. Sun YH, Fang R, Gao B, Han XK, Zhang JH, Pao W, et al. Comparable rate of EGFR kinase domain mutation in lung adenocarcinomas from Chinese male and female never-smokers. Acta Pharmacol Sin. 2010;31: 647–648. pmid:20418893
- 12. Rosell R, Moran T, Queralt C, Porta R, Cardenal F, Camps C, et al. Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med. 2009;361: 958–967. pmid:19692684
- 13. Shi Y, Au JS, Thongprasert S, Srinivasan S, Tsai CM, Khoa MT, et al. A prospective, molecular epidemiology study of EGFR mutations in Asian patients with advanced non-small-cell lung cancer of adenocarcinoma histology (PIONEER). J Thorac Oncol. 2014;9: 154–162. pmid:24419411
- 14. Fukuoka M, Wu YL, Thongprasert S, Sunpaweravong P, Leong SS, Sriuranpong V, et al. Biomarker analyses and final overall survival results from a phase III, randomized, open-label, first-line study of gefitinib versus carboplatin/paclitaxel in clinically selected patients with advanced non-small-cell lung cancer in Asia (IPASS). J Clin Oncol. 2011;29: 2866–2874. pmid:21670455
- 15. Chou TY, Chiu CH, Li LH, Hsiao CY, Tzen CY, Chang KT, et al. Mutation in the tyrosine kinase domain of epidermal growth factor receptor is a predictive and prognostic factor for gefitinib treatment in patients with non-small cell lung cancer. Clin Cancer Res. 2005;11: 3750–3757. pmid:15897572
- 16. Marchetti A, Martella C, Felicioni L, Barassi F, Salvatore S, Chella A, et al. EGFR mutations in non-small-cell lung cancer: analysis of a large series of cases and development of a rapid and sensitive method for diagnostic screening with potential implications on pharmacologic treatment. J Clin Oncol. 2005;23: 857–865. pmid:15681531
- 17. Rekhtman N, Paik PK, Arcila ME, Tafe LJ, Oxnard GR, Moreira AL, et al. Clarifying the spectrum of driver oncogene mutations in biomarker-verified squamous carcinoma of lung: lack of EGFR/KRAS and presence of PIK3CA/AKT1 mutations. Clin Cancer Res. 2012;18: 1167–1176. pmid:22228640
- 18. Li H, Pan Y, Li Y, Li C, Wang R, Hu H, et al. Frequency of well-identified oncogenic driver mutations in lung adenocarcinoma of smokers varies with histological subtypes and graduated smoking dose. Lung Cancer. 2013;79: 8–13. pmid:23098378
- 19. Na II, Park JH, Choe du H, Lee JK, Koh JS. Association of epidermal growth factor receptor mutations with metastatic presentations in non-small cell lung cancer. ISRN Oncol. 2011;2011: 756265. pmid:22091430
- 20. Su KY, Chen HY, Li KC, Kuo ML, Yang JC, Chan WK, et al. Pretreatment epidermal growth factor receptor (EGFR) T790M mutation predicts shorter EGFR tyrosine kinase inhibitor response duration in patients with non-small-cell lung cancer. J Clin Oncol. 2012;30: 433–440. pmid:22215752
- 21. Rosell R, Molina MA, Costa C, Simonetti S, Gimenez-Capitan A, Bertran-Alamillo J, et al. Pretreatment EGFR T790M mutation and BRCA1 mRNA expression in erlotinib-treated advanced non-small-cell lung cancer patients with EGFR mutations. Clin Cancer Res. 2011;17: 1160–1168. pmid:21233402
- 22. Yasuda H, Kobayashi S, Costa DB. EGFR exon 20 insertion mutations in non-small-cell lung cancer: preclinical data and clinical implications. Lancet Oncol. 2012;13: e23–31. pmid:21764376
- 23. Oxnard GR, Lo PC, Nishino M, Dahlberg SE, Lindeman NI, Butaney M, et al. Natural history and molecular characteristics of lung cancers harboring EGFR exon 20 insertions. J Thorac Oncol. 2013;8: 179–184. pmid:23328547
- 24. Sasaki H, Endo K, Takada M, Kawahara M, Kitahara N, Tanaka H, et al. EGFR exon 20 insertion mutation in Japanese lung cancer. Lung Cancer. 2007;58: 324–328. pmid:17686547
- 25. Arcila ME, Nafa K, Chaft JE, Rekhtman N, Lau C, Reva BA, et al. EGFR exon 20 insertion mutations in lung adenocarcinomas: prevalence, molecular heterogeneity, and clinicopathologic characteristics. Mol Cancer Ther. 2013;12: 220–229. pmid:23371856