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Originally published as JCO Early Release 10.1200/JCO.2005.04.3224 on February 27 2006 © 2006 American Society of Clinical Oncology. Use of Cigarette-Smoking History to Estimate the Likelihood of Mutations in Epidermal Growth Factor Receptor Gene Exons 19 and 21 in Lung Adenocarcinomas
From the Departments of Surgery, Medicine, Epidemiology and Biostatistics, and Pathology, Memorial Sloan-Kettering Cancer Center; Weill Medical College of Cornell University, New York, NY; and the Genome Sequencing Center, Washington University, St Louis, MO. Address reprint requests to Mark G. Kris, MD, Memorial Hospital, 1275 York Avenue, New York, NY 10021
PURPOSE: Lung adenocarcinomas with mutations in exons 19 and 21 of the epidermal growth factor receptor gene (EGFR) demonstrate sensitivity to gefitinib or erlotinib. Investigators have reported an association between EGFR mutations and the amount and duration of cigarette smoking, with the highest incidence of mutations seen in never smokers.
METHODS: EGFR exon 19 and 21 mutation status was determined in 265 tumor samples using direct sequencing, polymerase chain reaction (PCR), or PCR-based restriction fragment length polymorphism analysis. A detailed smoking history was obtained. Patients were categorized as never smokers (< 100 lifetime cigarettes), former smokers (quit RESULTS: We detected EGFR mutations in 34 (51%) of 67 never smokers (95% CI, 38% to 64%), 29 (19%) of 151 former smokers (95% CI, 13% to 27%), and two (4%) of 47 current smokers (95% CI, 1% to 16%). Significantly fewer EGFR mutations were found in people who smoked for more than 15 pack-years (P < .001) or stopped smoking less than 25 years ago (P < .02) compared with individuals who never smoked. The number of smoking pack-years and smoke-free years predicted the prevalence of EGFR mutations (areas under receiver operating characteristic curve = 0.78 and 0.77, respectively). CONCLUSION: The likelihood of EGFR mutations in exons 19 and 21 decreases as the number of pack-years increases. Mutations were less common in people who smoked for more than 15 pack-years or who stopped smoking cigarettes less than 25 years ago. These data can assist clinicians in assessing the likelihood of exon 19 and 21 EGFR mutations in patients with lung adenocarcinoma when mutational analysis is not feasible.
Mutations in the epidermal growth factor receptor (EGFR) gene have been identified in patients with lung adenocarcinomas.1-3 In the initial reports and in most, but not all, follow-up reports, these EGFR mutations have been associated with response to erlotinib or gefitinib.4-12 Two classes of EGFR mutations, exon 19 deletions and exon 21 L858R substitutions, are the most frequent mutations, representing 85% to 90% of EGFR mutations reported. EGFR exon 19 deletions and exon 21 L858R substitutions have also been the mutations best correlated with response to gefitinib or erlotinib. The presence of EGFR mutations has also been associated with treatment in East Asian countries, female sex, bronchioloalveolar carcinoma subtype of adenocarcinoma, and improved survival.5,7,13 One factor with a very strong association with these sensitivity mutations was a history of never smoking cigarettes.5,14-16 Patients with a high likelihood of response to treatment with tyrosine kinase inhibitors can be identified by molecular analysis of lung tumor tissue to detect these sensitivity mutations. Acquiring adequate tissue for EGFR mutational analysis, however, is often not feasible before the initiation of treatment, particularly in patients with advanced disease who are diagnosed on the basis of cytologic material. The goal of this study was to determine the elements of a patient's smoking history that predict the presence or absence of EGFR mutations.
Patients and Data Collection We evaluated 265 patients with adenocarcinoma of the lung seen at Memorial Sloan-Kettering Cancer Center (MSKCC; New York, NY). Specimens came from three sources: fresh-frozen tumors from a lung cancer tumor bank of resected lung cancers at MSKCC, a database of patients treated with gefitinib or erlotinib, and tumor specimens submitted to the MSKCC Laboratory of Diagnostic Molecular Pathology for EGFR mutational analysis.
Detailed smoking histories including age at first cigarette, packs per day, and number of smoking and smoke-free years (after quitting) were obtained. Patients were categorized as never smokers (< 100 lifetime cigarettes), former smokers (quit
EGFR Mutational Analysis
Statistical Analysis
Lung adenocarcinomas from 265 patients (176 women, 89 men) were analyzed for mutations in EGFR exon 19 and exon 21. EGFR mutations were detected in 65 tumors (25%). Mutations were seen in 40 women (23%) and 24 men (27%; P = .55). EGFR mutations were identified in never smokers (34 [51%] of 67; 95% CI, 38% to 64%), former smokers (29 [19%] of 151; 95% CI, 13% to 24%), and current smokers (2 [4%] of 47; 95% CI, 1% to 16%; Fig 1). The incidence of mutations in exon 19 or exon 21 was similar for men and women.
Never versus current and former smokers (P < .001), years smoked (P < .001), years smoke free (P = .004), and total pack-years (P < .001) were associated with the presence of an EGFR mutation by Fisher's exact and the Wilcoxon rank sum tests. Age at first cigarette (P = .46) was not associated with EGFR mutation. The incidence of EGFR mutations decreased with increasing number of pack-years of cigarette smoking. Patients with smoking histories of 16 to 75 pack-years had a 10% incidence of EGFR mutations compared to 46% among individuals smoking for 15 or fewer pack-years (P < .001). No EGFR mutations were detected in the 28 patients who smoked for more than 75 pack-years. EGFR mutations were detected in 51% of never smokers and in 37%, 46%, and 30% of patients with smoking histories of 1 to 5, 6 to 10, and 11 to 15 pack-years, respectively (Table 1). These data were similar for exon 19 and 21 mutations considered separately.
The presence of EGFR mutations by smoke-free years is listed in Table 2. The incidence of mutations in EGFR for zero to 25 smoke-free years was 8%; for patients who were smoke free for more than 25 years, the incidence of EGFR mutations was 40% (P < .001).
The area under the ROC curve for smoking history parameters are 0.72 for smoking status at diagnosis, 0.72 for age at first cigarette, 0.74 for number of packs per day, 0.77 for years smoke free, 0.78 for number of years smoked, and 0.78 for total pack-years. Among the three parameters with the largest area under the curve, total pack-years and number of years smoked are highly correlated. We chose to derive a predictor for EGFR mutations based on total pack-years and number of years smoke free. A smoking history of less than 15 total pack-years was 82% sensitive and 70% specific for predicting the presence of EGFR mutations (Fig 2A). Similarly, a total of more than 25 smoke-free years was 78% sensitive and 71% specific for EGFR mutations (Fig 2B). The categories of less than 15 total pack-years or more than 25 smoke-free years had 89% sensitivity and 62% specificity. Without cross validation, these sensitivity and specificity estimates may overestimate the accuracy of these predictors.
Molecular features can identify patients likely to respond to targeted therapies. At least 12 published series have associated partial response rates to tyrosine kinase inhibitors to the presence of mutations in EGFR.1-8,10,18-20 The presence of these mutations has since been associated with a survival advantage as well, both in patients who have never received EGFR tyrosine kinase inhibitors and in patients treated with gefitinib,5,20 suggesting that molecular characteristics are clinically relevant for patients with adenocarcinoma of the lung. The techniques currently used to identify patients with a particular molecular abnormality, such as gene mutations or alterations in gene copy number, require the availability of adequate tumor specimens. The identification of clinical parameters that can serve as surrogates for a specific molecular characteristic, in this case a gene mutation, may prove useful, especially in the treatment of advanced lung cancer, where only small amounts of tissue are available. Mutations in EGFR that confer sensitivity to treatment with tyrosine kinase inhibitors are significantly associated with a never-smoking history.1,3,13,16 These sensitivity mutations have been detected in former smokers as well, and responses to gefitinib and erlotinib have also been documented in these patients, although they occur less frequently. We set out to discover whether amount or duration of cigarette smoking was associated with a likelihood of mutation in EGFR, which in turn predicts responsiveness to tyrosine kinase inhibitors of EGFR. To do this, we studied the smoking histories of patients with lung adenocarcinoma and its association with mutations in EGFR. We found that patients with a cigarette smoking history of more than 15 pack-years, or those who quit smoking less than 25 years ago, have a lower likelihood of harboring EGFR mutations compared with those persons who have never smoked cigarettes. A group of 51 patients (19% of the total studied) with limited cigarette exposure (<15 pack-years) had an incidence of EGFR mutations comparable to never smokers. Thus, treatment with tyrosine kinase inhibitors should not be limited to never smokers, but routinely expanded to former smokers with exposures of less than 15 pack-years or who are more than 25 years smoke free. By identifying only patients with specific mutations in EGFR exon 19 or with exon 21 L858R substitutions, our analysis did not include less common EGFR mutations. The preponderance of the clinical data associating clinical response to gefitinib or erlotinib with EGFR mutations is based on the more common exon 19 deletions and exon 21 L858R substitutions. The clinical and biologic significance of other EGFR mutations (eg, mutations of L861, S768, E709, G719, and H835) remains unclear. In our analysis, the frequency of EGFR mutations in patients who had never smoked cigarettes was 51% (34 of 67 patients). The frequency of EGFR mutations has been observed to vary from 26% to 68% based on how the mutational analysis was performed, geography and other clinical characteristics of the patients investigated.5,12,13,16,18,21 Shigematsu et al16 reported the results of EGFR mutational analysis of tumor specimens from a geographically and ethnically diverse group of patients. The frequency of EGFR mutations in never smokers ranged from 27% for patients from the United States to 60% for patients from Japan. When combining all geographic and ethnic groups, however, Shigematsu et al identified EGFR mutations in 51% of never smokers (85 of 166), results very similar to ours. Clinical parameters that are associated with molecular determinants of treatment sensitivity or resistance can permit the more widespread application of therapeutic advances based on molecular characteristics. This approach is particularly relevant to lung cancer, in which the majority of individuals with advanced disease at diagnosis have insufficient pathologic material for EGFR sequencing or other molecular studies. The data presented here demonstrate that a universally available and verifiable clinical characteristic can estimate the incidence of mutations in exons 19 and 21 of EGFR, and assist the clinician in choosing therapy for a specific patient.
The authors indicated no potential conflicts of interest.
EGFR (epidermal growth factor receptor): Also known as HER-1, EGFR belongs to a family of receptors (HER-2, HER-3, HER-4 are other members of the family) and binds to the EGF, TGF-  , and other related proteins, leading to the generation of proliferative and survival signals within the cell. It also belongs to the larger family of tyrosine kinase receptors and is generally overexpressed in several solid tumors of epithelial origin. Exon: Segment of a gene that consists of a sequence of nucleotides that encodes amino acids in the protein. Genes are often made up of multiple exons separated by introns that do not encode amino acids in the protein. Pack-year: A unit to quantify exposure to cigarette smoke. A pack-year is calculated by multiplying the number of packs of cigarettes smoked per day by the number of years smoked. Receiver operating characteristic (ROC) curve: ROC curves plot the true positive rate (sensitivity) against the false-positive rate (1-specificity) for different cutoff levels of a test. The area under the curve is a measure of the accuracy of the test. An area of 1.0 represents a perfect test (all true positives), whereas an area of 0.5 represents a worthless test.
Supported in part by Genentech Inc, the Steps for Breath Lung Cancer Research Program of the Society of Memorial Sloan-Kettering, and the Carmel Hill Fund. Presented in part at the 41st Annual Meeting of the American Society of Clinical Oncology, Orlando, FL, May 13-17, 2005. Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
1 year ago), or current smokers (quit < 1 year ago). 
