Background: The EML4-ALK fusion gene is more frequently found in younger, never smoking patients with lung cancer. Meanwhile, never smokers exposed to secondhand tobacco smoke (SHS) during childhood are diagnosed at a younger age compared with never smoking patients with lung cancer who are not exposed. We, therefore, hypothesized that SHS, which can induce DNA damage, is associated with the EML4-ALK fusion gene.

Methods: We compared the frequency of the EML4-ALK fusion gene among 197 never smoker patients with lung cancer with and without a history of exposure to SHS during childhood at Mayo Clinic.

Results: The EML4-ALK fusion gene was detected in 33% of cases from never smokers with a history of SHS exposure during childhood, whereas 47% of never smoking lung cancer cases without a history of childhood SHS exposure tested positive for the fusion gene.

Conclusions: The EML4-ALK fusion gene is not enriched in tumors from individuals exposed to SHS during childhood.

Impact: These data suggest that childhood exposure to SHS is not a significant etiologic cause of the EML4-ALK fusion gene in lung cancer. Cancer Epidemiol Biomarkers Prev; 23(7); 1432–4. ©2014 AACR.

Although they were initially thought to be common only to hematologic malignancies, fusion genes caused by chromosomal rearrangements are a now recognized as a feature of solid malignancies. The anaplastic lymphoma kinase (ALK)/echinoderm microtubule-associated protein-like 4 (EML4) fusion gene was recently identified in non–small cell lung cancer (1). It is caused by an inversion of chromosome 2 and is more frequently detected in younger, never smoking patients (2). EML4-ALK–positive lung cancer represents approximately 5% of non–small cell lung cancer and is very sensitive to ALK inhibitors such as crizotinib (3). We (4), and others (5), previously reported that childhood exposure to secondhand tobacco smoke (SHS) is associated with a higher risk of lung cancer among adults, and that never smoker patients exposed to SHS during childhood, like patients with EML4-ALK fusion genes, are 7 to 15 years younger at the time of their lung cancer diagnosis compared with patients without a history of childhood SHS exposure (4). Because SHS can induce DNA damage (6), we tested the hypothesis that exposure to SHS during childhood was a potential cause of EML4-ALK formation in lung cancer.

Study design, sampling, and data collection

The 300 patient samples included in this study were selected from the Mayo Clinic Lung Cancer Cohort, an observational follow-up study (4, 7). All patients were never-smokers, defined as having smoked zero to 99 cigarettes during their lifetime. Secondhand smoke exposure data were collected via questionnaire and available for 197 of 300 participants. Specific information was also gathered on whether exposure was from a parent, spouse, or coworker; the number of cigarettes per day they were exposed to; and the number of years for which they were exposed.

EML4-ALK positivity was determined previously using two widely used and accepted assays (8). First, FISH for the ALK locus rearrangement was conducted using an interphase molecular cytogenetic assay and commercially available ALK probe (Vysis; ref. 8). Samples were considered positive if 15% or more of at least 100 cells counted showed splitting of the florescent probes flanking the ALK locus. ALK1 protein expression, commonly considered as a surrogate for EML4-ALK positivity, was detected by IHC using an ALK1 monoclonal antibody (Dako), as described previously (7). An IHC score was assigned to each case using the following criteria: tumor section contained ≥10% tumor cells; score of 3 intense, granular cytoplasmic staining; score of 2, moderate, smooth cytoplasmic staining; score of 1, faint cytoplasmic staining; and score of 0, no staining (7).

For this study, EML4-ALK positivity was defined as a positive FISH score and IHC score of 2 or 3. EML4-ALK–negative samples were defined as FISH negative and an IHC score of 0 or 1. All FISH and ALK IHC interpretation was performed without the knowledge of IHC results for ALK and vice versa.

Thirty-four of the 300 (11.1%) patients included in this study were positive for EML4-ALK, which was consistent with previous reports of never smokers. Among this cohort, data on exposure to SHS during childhood were available for 197 of participants. We compared the frequency of childhood SHS exposure between those positive (33%) and negative (47.1%) for EML4-ALK using a χ2 test, but did not find evidence to reject the null hypothesis, i.e., we did find an enrichment of EML4-ALK–positive tumors among those with a history of childhood exposure to SHS (P = 0.183; Table 1). We further tested whether the duration of exposure, dose, or source of exposure (mother versus father) was associated with EML4-ALK status, but did not find any statistically significant relationships (Table 1). Exposure to secondhand smoke during adulthood was not associated with the presence of the EML4-ALK fusion gene (Table 1).

Table 1.

Characteristics of ALK-positive and ALK-negative childhood exposure to SHS

Positive (N = 34)Negative (N = 266)Total (N = 300)P
Any childhood ETS 27 170 197 0.1831 
 1 = Yes 9 (33.3%) 80 (47.1%) 89 (45.2%)  
 0 = No 18 (66.7%) 90 (52.9%) 108 (54.8%)  
Years of childhood ETS    0.6233 
 N 66 75  
 Mean (SD) 19.8 (7.8) 25.1 (18.0) 24.5 (17.2)  
 Median 19.0 19.0 19.0  
 Q1, Q3 16.0, 19.0 17.0, 28.0 17.0, 28.0  
Est. pack-years child ETS    0.4359 
 N 62 71  
 Mean (SD) 15.3 (10.6) 23.5 (22.2) 22.5 (21.2)  
 Median 12.0 16.8 16.5  
 Q1, Q3 9.0, 28.5 9.0, 27.0 9.0, 27.0  
When exposed to cig ETS 27 175 202 0.0242 
 Unknown 0 (0.0%) 5 (0.0%) 5 (0.0%)  
 0 = No ETS 12 (44.4%) 31 (18.2%) 43 (21.8%)  
 1 = Child ETS 3 (11.1%) 25 (14.7%) 28 (14.2%)  
 2 = Adult ETS 6 (22.2%) 59 (34.7%) 65 (33.0%)  
 3 = Life ETS 6 (22.2%) 55 (32.4%) 61 (31.0%)  
Any father ETS 27 175 202 0.3500 
 1 = Yes 9 (33.3%) 75 (42.9%) 84 (41.6%)  
 0 = No 18 (66.7%) 100 (57.1%) 118 (58.4%)  
Amount of father ETS 27 175 202 0.4634 
 0 = None 18 (66.7%) 104 (59.4%) 122 (60.4%)  
 1 = Light 3 (11.1%) 20 (11.4%) 23 (11.4%)  
 2, 3 = Moderate to heavy 6 (22.2%) 51 (29.1%) 57 (28.2%)  
Any mother ETS 27 175 202 0.3390 
 1 = Yes 1 (3.7%) 21 (12.0%) 22 (10.9%)  
 0 = No 26 (96.3%) 154 (88.0%) 180 (89.1%)  
Amount of mother ETS 27 175 202 0.2852 
 0 = None 26 (96.3%) 156 (89.1%) 182 (90.1%)  
 1 = Light 1 (3.7%) 2 (1.1%) 3 (1.5%)  
 2, 3 = Moderate to heavy 0 (0.0%) 17 (9.7%) 17 (8.5%)  
Positive (N = 34)Negative (N = 266)Total (N = 300)P
Any childhood ETS 27 170 197 0.1831 
 1 = Yes 9 (33.3%) 80 (47.1%) 89 (45.2%)  
 0 = No 18 (66.7%) 90 (52.9%) 108 (54.8%)  
Years of childhood ETS    0.6233 
 N 66 75  
 Mean (SD) 19.8 (7.8) 25.1 (18.0) 24.5 (17.2)  
 Median 19.0 19.0 19.0  
 Q1, Q3 16.0, 19.0 17.0, 28.0 17.0, 28.0  
Est. pack-years child ETS    0.4359 
 N 62 71  
 Mean (SD) 15.3 (10.6) 23.5 (22.2) 22.5 (21.2)  
 Median 12.0 16.8 16.5  
 Q1, Q3 9.0, 28.5 9.0, 27.0 9.0, 27.0  
When exposed to cig ETS 27 175 202 0.0242 
 Unknown 0 (0.0%) 5 (0.0%) 5 (0.0%)  
 0 = No ETS 12 (44.4%) 31 (18.2%) 43 (21.8%)  
 1 = Child ETS 3 (11.1%) 25 (14.7%) 28 (14.2%)  
 2 = Adult ETS 6 (22.2%) 59 (34.7%) 65 (33.0%)  
 3 = Life ETS 6 (22.2%) 55 (32.4%) 61 (31.0%)  
Any father ETS 27 175 202 0.3500 
 1 = Yes 9 (33.3%) 75 (42.9%) 84 (41.6%)  
 0 = No 18 (66.7%) 100 (57.1%) 118 (58.4%)  
Amount of father ETS 27 175 202 0.4634 
 0 = None 18 (66.7%) 104 (59.4%) 122 (60.4%)  
 1 = Light 3 (11.1%) 20 (11.4%) 23 (11.4%)  
 2, 3 = Moderate to heavy 6 (22.2%) 51 (29.1%) 57 (28.2%)  
Any mother ETS 27 175 202 0.3390 
 1 = Yes 1 (3.7%) 21 (12.0%) 22 (10.9%)  
 0 = No 26 (96.3%) 154 (88.0%) 180 (89.1%)  
Amount of mother ETS 27 175 202 0.2852 
 0 = None 26 (96.3%) 156 (89.1%) 182 (90.1%)  
 1 = Light 1 (3.7%) 2 (1.1%) 3 (1.5%)  
 2, 3 = Moderate to heavy 0 (0.0%) 17 (9.7%) 17 (8.5%)  

Abbreviation: ETS, environmental tobacco smoke.

In recent years, the discovery of fusion genes in cancer has accelerated, in part due to the advent of next-generation sequencing technologies. These discoveries have improved outcomes for many patients, given that many are potent driver oncogenes and sensitive to targeted therapies. However, the etiologic cause of such fusion genes, for the most part, remains unknown. In this study, we hypothesized that the EML4-ALK fusion gene is caused by exposure to SHS during childhood. We based this hypothesis on the observation that (i) patients with lung cancer with the EML4-ALK fusion gene and those exposed to SHS during childhood are both diagnosed with lung cancer at a younger age, and (ii) exposure to SHS can cause DNA damage. We did not find statistical evidence that the EML4-ALK is associated with a history of SHS exposure during childhood. Potential limitations of this study include the possibility of recall bias among those reporting childhood SHS exposure. Although it is possible that other fusion genes could be caused by childhood exposure to SHS, our data do not support the hypothesis that the EML4-ALK fusion gene is associated with SHS exposure during childhood.

No potential conflicts of interest were disclosed.

Conception and design: B.M. Ryan, Y. Wang, J. Jen, E.S. Yi, P. Yang, C.C. Harris

Development of methodology: Y. Wang

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Wang, P. Yang

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): B.M. Ryan, Y. Wang, S. Olivo-Marston, P. Yang, C.C. Harris

Writing, review, and/or revision of the manuscript: B.M. Ryan, Y. Wang, J. Jen, S. Olivo-Marston, P. Yang, C.C. Harris

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Wang

Study supervision: B.M. Ryan, E.S. Yi, C.C. Harris

This work was supported by the Intramural Program of the Centre for Cancer Research, National Cancer Institute, NIH grants NIH-R01-CA80127, NIH-R01-CA84354, and NIH-R01-CA115857 (all to P. Yang). J. Jen is a recipient of the New Investigator Award from the American Cancer Society and supported by funding from Mayo Clinic Cancer Center and the Center for Individualized Medicine. P. Yang, J. Jen, E.S. Yi, and Y. Wang received support from The Mayo Clinic Foundation.

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