Abstract
Background
Lung cancer patients with mutations in EGFR tyrosine kinase have improved prognosis when treated with EGFR inhibitors. We hypothesized that EGFR mutations may be related to residential radon or passive tobacco smoke.
Methods
This hypothesis was investigated by analyzing EGFR mutations in seventy lung tumors from a population of never and long-term former female smokers from Missouri with detailed exposure assessments. The relationship with passive-smoking was also examined in never-smoking female lung cancer cases from the Mayo clinic.
Results
Overall, the frequency of EGFR mutation was 41% [95% Confidence Interval (CI): 32-49%]. Neither radon nor passive-smoking exposure was consistently associated with EGFR mutations in lung tumors.
Conclusions
The results suggest that EGFR mutations are common in female, never-smoking, lung cancer cases from the U.S, and EGFR mutations are unlikely due to exposure to radon or passive-smoking.
Keywords: EGFR mutations, never-smokers, lung cancer, radon, passive-smoking, second hand smoke, tobacco smoke
Introduction
Among never-smokers, lung cancer is the seventh leading cause of cancer death. A large proportion of lung cancer in never-smokers remains unexplained by established environmental risk factors. However, radon and passive-smoke exposure were associated with lung cancer in never-smokers in several studies (for review (1)).
Lung cancer has a poor prognosis overall, but small molecule inhibitors of EGFR result in improved survival in some patients. Therapeutic response correlates with somatic mutations in the EGFR gene. Those mutations are inversely correlated with cigarette smoking and more frequently observed in never-smokers (for review (2)). We investigated the possibility that residential radon or passive-smoking was associated with the presence of EGFR mutations in lung tumors in two populations of female never and long-term former smokers.
Methods
Study Populations
The Missouri Women’s Health Study case series included Caucasian lung cancer cases nested within a case-control study of never- and former-smoking women (3-4). Lung cancer patients from the Mayo Clinic were described previously (5). Cases were limited to Caucasian women to ensure comparability to the Missouri study.
EGFR Mutation Analysis
Missouri Women
DNA previously isolated from microdissected tumor samples (4), available from 105 of 132 samples, was used for EGFR mutation analysis in the Laboratory of Human Carcinogenesis. Due to evaporation, the majority of DNA samples (74% or 78/105) were reconstituted using 10 μl of Tris-EDTA buffer (pH 7.5). PCR amplification was performed as a 50 μl reaction including 2 μl DNA stock solution, 1.25 U of Native Pfu DNA polymerase (Stratagene), 1X Pfu buffer, 300 nM forward and reverse primers for either exon 19 or exon 21 of EGFR; primers were identical to those reported previously (6). Amplification was performed using the following conditions: 95°C for 5 minutes followed by 40 cycles of 96°C for 45 sec, 58°C for 1 min, and 72°C for 1 min; a terminal extension cycle of 5 min at 72°C was included. If initial PCR reactions failed to amplify, a second PCR amplification reaction was performed using 5 μl of a 1:10 dilution of the PCR reaction mixture. Samples that failed the first series of amplifications were re-amplified using a second aliquot of genomic DNA. Overall, thirty-two (30%) of 105 genomic samples failed to amplify. DNA sequencing was performed as per manufacturer’s instructions on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems) by NCI DNA MiniCore Facility using the PCR amplification primers. Forward and reverse sequences were 100% concordant.
Mayo Clinic
EGFR mutations were analyzed at the Mayo Clinic as part of oncogene mutation screening using the OncoCarta™ Panel v1.0 (Sequenom, San Diego, CA) on the Sequenom MassArray Genetic Analysis platform following manufacturer’s protocol. Data analysis was performed using MassArray Typer Analyzer 4.0 software (Sequenom, San Diego, CA). Performance of the assay was evaluated against a panel of lung tumor samples and cell lines with previously identified mutations. EGFR gene mutation data was available for all 73 cases from the Mayo clinic study.
Statistical Analysis
Samples with incomplete sequencing data for EGFR (e.g. failed amplification at one or more exons) were considered missing. The Mayo Clinic study had mutation data on additional EGFR exons compared to the Missouri study. Cases with mutations in exons other than 19 and 21 were considered wild-type for EGFR (N=3). Results were similar when they were considered mutant (data not shown).
Never-smokers were defined as persons who had either smoked < 100 cigarettes or not used any tobacco products during their lifetimes. To examine association of any exposure to passive-smoking in the Missouri study, categories of exposure (< 21, 21-52, and >52 pack-years) were combined and compared to 0 pack-years. Former-smokers in the Missouri study abstained from tobacco for at least 15 years prior to interview (3). In the Mayo Clinic Study, passive-smoke dosimetry included both adult and/or in childhood exposures. Data analysis was performed by SAS version 9.1 (SAS Institute, Cary, NC) using two-sided tests in the Laboratory of Human Carcinogenesis.
Results
Twenty-four of the Missouri cases (34%; 95% CI: 23% - 47%) and thirty-four of the Mayo clinic cases (47%; 95% CI: 35% - 59%) had mutations detected in exons 19 or 21 of EGFR (Table 1). Overall, the mutation frequency was 41% (95% CI: 32% - 49.0%).
Table 1.
EGFR Gene Mutations in Never and Former Smoking Lung Cancer Patients
| Patient ID | Histology | EGFRexon | Mutation Type | Codon(s) | Nucleotide Change |
Amino Acid Change |
|---|---|---|---|---|---|---|
| Missouri Women | ||||||
| 2 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 6 | other/mixed | 21 | Point | 858 | CTG>CGG | Leu>Arg |
| 48 | small cell carcinoma | 21 | Point | 866 | GAG>GAT | Glu>Asp |
| 54 | adenocarcinoma | 21 | Point | 858 | CTG>CGG | Leu>Arg |
| 56 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 57 | adenocarcinoma | 21 | Point | 858 | CTG>CGG | Leu>Arg |
| 60 | adenocarcinoma | 21 | Point | 858 | CTG>CGG | Leu>Arg |
| 69 | adenocarcinoma | 21 | Point | 858 | CTG>CGG | Leu>Arg |
| 71 | adenocarcinoma | 21 | Point | 858 | CTG>CGG | Leu>Arg |
| 72 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 74 | adenocarcinoma | 21 | Point | 833 | TTG>TTT | Leu>Phe |
| 76 | adenocarcinoma | 19 | Deletion plus insertion | del Leu747-Pro753 (ins Ser)a | ||
| 78 | adenocarcinoma | 19 | Deletion plus insertion | del Leu747-Ala750 (ins Pro)a | ||
| 81 | adenocarcinoma | 19 | Point | 743 | GCT>ACT | Ala>Thr |
| 88 | adenocarcinoma | 21 | Point (silent) | 858 (silent) | CTG>CTT | Leu>Leu |
| 95 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 104 | adenocarcinoma | 21 | Point | 864 | GCG>GTG | Ala>Val |
| 105 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 126 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 129 | bronchioloalveolar carcinoma |
19 | Deletion | del Glu746-Ala750 | ||
| 134 | adenocarcinoma | 21 | Point | 858 | CTG>CGG | Leu>Arg |
| 135 | bronchioloalveolar carcinoma |
19 | Deletion plus insertion | del Glu746-Ser752 (ins Val)a | ||
| 136 | adenocarcinoma | 21 | Point (silent) | 848 (silent) | CCG>CCT | Pro>Pro |
| 139 | other/mixed | 19 | Deletion | del Ser752 -Ile759 | ||
|
| ||||||
| Mayo Clinic Study | ||||||
| adenosquamous | ||||||
| 921901995 | carcinoma | 21 | Point | 858 | CTG>CGG | Leu>Arg |
| 921901997 | adenocarcinoma | 21 | Point | 858 | CTG>CGG | Leu>Arg |
| 921902006 | bronchioloalveolar carcinoma |
21 | Point | 858 | CTG>CGG | Leu>Arg |
| 921902011 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 921902017 | adenosquamous carcinoma |
21 | Point | 858 | CTG>CGG | Leu>Arg |
| 921902018 | adenocarcinoma bronchioloalveolar |
19 | Deletion | del Glu746-Ala750 | ||
| 921902021 | carcinoma | 19 | Deletion plus insertion | del Leu747-Pro753 (ins Ser)a | ||
| 921902023 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 921902024 | adenocarcinoma | 21 | Point | 858 | CTG>CGG | Leu>Arg |
| 921902032 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 921902033 | adenosquamous carcinoma |
19 | Deletion | del Glu746-Ala750 | ||
| 921902049 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 921902055 | adenocarcinoma | 21 | Point | 858 | CTG>CGG | Leu>Arg |
| 921902063 | adenocarcinoma | 19 | Deletion plus insertion | del Leu747-Ala750 (ins Pro)a | ||
| 921902067 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 921902070 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 921902071 | adenocarcinoma | 19 | Deletion plus insertion | del Glu746-Thr751 (ins Val)a | ||
| 921902084 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 921902086 | bronchioloalveolar carcinoma |
19 | Deletion | del Glu746-Ala750 | ||
| 921902118 | adenosquamous carcinoma |
21 | Point | 858 | CTG>CGG | Leu>Arg |
| 921902119 | adenocarcinoma | 21 | Point | 858 | CTG>CGG | Leu>Arg |
| 921902121 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 921902132 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 921902140 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 921902148 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 921902154 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 921902157 | squamous cell carcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 921902160 | squamous cell carcinoma | 19 | Deletion | del Glu746-Ala750 | ||
| 921902161 | adenocarcinoma | 21 | Point | 858 | CTG>CGG | Leu>Arg |
| 921902163 | adenocarcinoma | 19 | Deletion plus insertion | del Leu747-Ala750 (ins Pro)a | ||
| 921902171 | adenocarcinoma | 21 | Point | 858 | CTG>CGG | Leu>Arg |
| 921902172 | adenocarcinoma | 21 | Point | 858 | CTG>CGG | Leu>Arg |
| 921902175 | adenocarcinoma | 21 | Point | 858 | CTG>CGG | Leu>Arg |
| 921902187 | adenocarcinoma | 19 | Deletion | del Glu746-Ala750 | ||
ins, Insertion of Amino Acid in parentheses
While there was a difference in the quartiles of radon exposure associated with EGFR mutation (P=0.01), this was not significant when exposure was dichotomized at the median (P=0.14, Fisher’s Exact Test) and no difference was observed when considering radon as a continuous measure (P=0.16) and there was no evidence for a dose-response relationship with radon exposure (Table 2).
Table 2.
Association of EGFR mutations with Patient Charactersitics
| Missouri Women EGFR |
Mayo Clinic Study EGFR |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| −Mutation N=46 |
+Mutation N=24 |
−Mutation N=39 |
+Mutation N=34 |
|||||||
| N | % | N | % | p-value | p-value | |||||
| Histological Subtype | ||||||||||
| Adenocarcinomaa | 34 | 74 | 19 | 80 | 0.98b | 28 | 72 | 29 | 85 | 0.28b |
| Bronchioloalveolar carcinoma | 4 | 9 | 2 | 8 | 4 | 10 | 3 | 9 | ||
| Squamous cell carcinoma | 2 | 4 | 0 | 0 | 3 | 8 | 2 | 6 | ||
| Small cell carcinoma | 1 | 2 | 1 | 4 | 0 | 0 | 0 | 0 | ||
| Other | 5 | 11 | 2 | 8 | 4 | 10 | 0 | 0 | ||
| Age (years) | ||||||||||
| median, IQR | 76 | 64-79 | 66 | 61-78 | 0.24c | 68 | 57-75 | 72 | 67-79 | 0.06c |
| missing | 3 | 1 | ||||||||
| Passive-Smoke | ||||||||||
| No exposure | 21 | 47 | 17 | 74 | 0.04b | 10 | 32 | 4 | 15 | 0.22b |
| Any exposure | 24 | 53 | 6 | 26 | 21 | 68 | 22 | 85 | ||
| Missing | 1 | 1 | 8 | 8 | ||||||
| Passive-Smoke (Pack-years) | ||||||||||
| No exposure | 21 | 47 | 17 | 74 | 0.08b | nd | nd | |||
| < 21 | 10 | 22 | 3 | 13 | ||||||
| 21-52 | 11 | 24 | 1 | 4 | ||||||
| >52 | 3 | 7 | 2 | 9 | ||||||
| Missing | 1 | 1 | ||||||||
| Passive-Smoke (Adult Exposure) | ||||||||||
| No exposure | nd | nd | 11 | 35 | 8 | 31 | 0.78b | |||
| Any exposure | 20 | 65 | 18 | 69 | ||||||
| Missing | 8 | 8 | ||||||||
| Passive-Smoke (Child Exposure) | ||||||||||
| No exposure | nd | nd | 22 | 71 | 13 | 50 | 0.17b | |||
| Any exposure | 9 | 29 | 13 | 50 | ||||||
| Missing | 8 | 8 | ||||||||
| Radon Exposure (Bq/m3) | ||||||||||
| 4.8 - 33.3 | 13 | 30 | 5 | 23 | 0.01b | nd | nd | |||
| 35.2 - 55.5 | 5 | 11 | 9 | 41 | ||||||
| 56.2 - 82.7 | 9 | 20 | 6 | 27 | ||||||
| > 82.7 | 17 | 39 | 2 | 9 | ||||||
| missing | 2 | 2 | ||||||||
| Radon Exposure (Bq/m3) | ||||||||||
| median, IQR | 63.7 | 30.5-94.1 | 46.5 | 37.0-57.4 | 0.16c | nd | nd | |||
| missing | 2 | |||||||||
Five of the adenocarcinomas in the Mayo Clinic Study were adenosquamous histology.
Fisher’s Exact test
Wilcoxon two-sample test
nd , not determined
In the Missouri Women’s Health Study cases, there was an inverse association of EGFR mutations with any exposure to passive-smoke, but no clear dose response relationship was observed with passive-smoke exposure quantified in pack-years. In the Mayo clinic population, no association was observed between EGFR mutations and adult exposure, childhood exposure, or any exposure to passive-smoke (Table 2).
Discussion
Our data do not support the hypothesis that radon exposure contributes to mutations in EGFR. The mutation frequency appeared elevated at low-dose exposure and diminished at higher exposure levels, but we noticed a similar trend with TP53 mutations (4). The relationship of radon with lung cancer risk is thought to be linear (1), so our inverse trend between radon dose and EGFR mutations probably occurred by chance.
We observed an inverse association of passive-smoke exposure with EGFR mutations in lung tumors in the Missouri study, but this finding failed to replicate in the Mayo Clinic never-smoker patient cohort. Previously, an inverse association with passive-smoke exposure as an adult or in childhood was observed (7). However, another study linked long-term exposure to passive-smoking with excess EGFR mutations (8).
In conclusion, we observed a high frequency of EGFR mutations in lung tumors from never-smoking and long-term former-smoking women in the U.S., but no association between EGFR mutations with passive-smoking or residential radon exposure.
Acknowledgements
We thank Karen MacPherson for bibliographic assistance.
Funding
Grant support: Intramural Research Program of the NIH, NCI and CCR. The support from the Mayo Clinic study was provided by the Mayo Foundation, Mayo Clinic Cancer Center, Center for Individualized Medicine and NIH grants CA-77118 (Yang) and CA-80127 (Yang). The Radiation Effects Research Foundation (RERF), Hiroshima and Nagasaki, Japan is a private, non-profit foundation funded by the Japanese Ministry of Health, Labour and Welfare (MHLW) and the U.S. Department of Energy (DOE), the latter in part through DOE Award DE-HS0000031 to the National Academy of Sciences. The views of the authors do not necessarily reflect those of the two governments.
Footnotes
Conflict of interest statement
No potential conflicts of interest were disclosed.
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