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(Chest. 2005;128:445-452.)
© 2005 American College of Chest Physicians

{alpha}1-Antitrypsin and Neutrophil Elastase Imbalance and Lung Cancer Risk*

Ping Yang, MD, PhD; William R. Bamlet, MS; Zhifu Sun, MD, PhD; Jon O. Ebbert, MD; Marie-Christine Aubry, MD; William R. Taylor, MS; Randolph S. Marks, MD; Claude Deschamps, MD, TS; Stephen J. Swensen, MD; Eric D. Wieben, PhD; Julie M. Cunningham, PhD; Lee Joseph Melton, MD and Mariza de Andrade, PhD

* From the Divisions of Epidemiology and Cancer Center (Drs. Yang, Sun, and Melton), Biostatistics (Mr. Bamlet and Dr. Andrade), Primary Care Internal Medicine (Dr. Ebbert), Anatomic Pathology (Dr. Aubry), Medical Oncology (Dr. Marks), and General Thoracic Surgery (Dr. Deschamps), the Genotyping Share Resource Laboratory (Mr. Taylor and Dr. Cunningham), the Department of Radiology (Dr. Swensen), and the Biochemistry and Molecular Biology and Genomics Research Center (Dr. Wieben), Mayo Clinic College of Medicine, Rochester, MN.

Correspondence to: Ping Yang, MD, PhD, Mayo Clinic College of Medicine, 200 First St SW, Rochester, MN 55905; e-mail: yang.ping{at}mayo.edu


   Abstract
TOP
 Abstract
Introduction
 Study Subjects and Methods
 Results
 Discussion
 References
 
Objective: Imbalance between {alpha}1-antitrypsin and neutrophil elastase is an underlying cause of lung tissue damage that may create a favorable host environment for carcinogenesis. We conducted a case-control study to investigate whether genetic variations indicative of {alpha}1-antitrypsin deficiency (A1ATD) or an excess of neutrophil elastase modify lung cancer risk

Design: The case patients were 305 consecutively identified primary lung cancer patients, and the control subjects were 338 community residents. Protease inhibitor-1 (PI1), encoding {alpha}1-antitrypsin, was typed by an isoelectric focusing assay. Neutrophil elastase-2 (ELA2), encoding neutrophil elastase, was typed by two single-nucleotide polymorphism sites. Multivariable logistic regression models tested the independent and interactive effects of PI1, ELA2, tobacco smoke exposure, COPD, and family history of lung cancer

Results: Sex and ethnicity were comparable between case patients and control subjects, but case patients were more likely to be smokers, and to have a history of COPD, environmental tobacco smoke exposure, and a positive family history of lung cancer. Haplotype analysis indicated an overall strong association between the two ELA2 markers and lung cancer risk. Our best-fitting model showed significant and independent effects of the PI1-deficient allele (odds ratio [OR], 2.0; 95% confidence interval [CI], 1.4 to 3.0) and the ELA2 T-G haplotype (OR, 4.1; 95% CI, 1.9 to 8.9) on lung cancer risk, and an increased risk (OR, 2.6; 95% CI, 2.4 to 2.8) for individuals carrying both a PI1-deficient allele and a G-G haplotype

Conclusions: Genotypes indicative of A1ATD and/or an excess of neutrophil elastase are significantly associated with lung cancer risk. Our findings may provide opportunities to better understand the mechanisms of lung cancer development and risk reduction.

Key Words: {alpha}1-antitrypsin deficiency • chronic obstructive • leukocyte elastase • lung neoplasm • pulmonary disease • tobacco smoking


   Introduction
TOP
 Abstract
 Introduction
Study Subjects and Methods
 Results
 Discussion
 References
 
While a majority of lung cancer patients have a history of tobacco use,12 the variation in lung cancer risk among smokers can be 20-fold.345 One well-documented host factor related to the risk of lung cancer is COPD, including emphysema and chronic bronchitis.67 COPD shares common etiologic factors with lung cancer, particularly cigarette smoking.8 Previous studies9 have suggested that {alpha}1-antitrypsin deficiency (A1ATD) not only can cause emphysema but is also associated with an increased risk of multiple malignancies, including lung cancer. Several mechanisms of tumorigenesis have been postulated between A1ATD and lung cancer development, as follows: excess neutrophil elastase, the counterpart of {alpha}1-antitrypsin, may facilitate cancer development by causing tissue damage and air trapping that fosters longer carcinogen exposure; may promote cancer progression by degrading the intercellular matrix barrier; and may lead to cancer development through the tumor necrosis factor signaling pathway.9 To test the hypothesis that {alpha}1-antitrypsin and neutrophil elastase may be critical in the causal pathway from tobacco smoke exposure to lung cancer development, we conducted a case-control study using functionally significant polymorphic markers to assess the role of protease inhibitor-1 (PI1) and neutrophil elastase-2 (ELA2), which encode functional variations of the two proteins in lung cancer risk in concert with known environmental and host factors.10


   Study Subjects and Methods
TOP
 Abstract
 Introduction
 Study Subjects and Methods
Results
 Discussion
 References
 
Study Participants and Data Collection
The research protocol was approved by the Mayo Clinic Institutional Review Board. Written informed consent was obtained from all subjects. As reported previously,101112 305 case patients were consecutively enrolled into the study from among patients who had received diagnoses and/or been treated for pathologically confirmed primary lung cancer at the Mayo Clinic between 1997 and 2001. Eligible patients were invited to participate in a baseline interview and a peripheral blood sample collection.

The 338 control subjects were frequency-matched to case patients by age, sex, and ethnic background from a pool of 2,335 Olmsted County, MN, residents who had attended the Mayo Clinic between 1998 and 2002, and who had a blood sample left over from their clinical tests.10 This design was chosen because Mayo Clinic is a major primary care provider for the local population, and > 90% of Olmsted County residents visit the Mayo Clinic at least once during any 3-year period.13 Eligible control subjects had no current or previously diagnosed malignancy (except nonmelanoma skin cancer) as of the date of phlebotomy. Control subjects received a self-administered questionnaire and a request for permission to use their leftover blood samples.

Data collected included information regarding each first-degree relative and the ancestral background of each subject’s paternal and maternal grandparents.11 The ethnic backgrounds of the case patients and control subjects were very similar, consisting mostly of white persons of non-Hispanic origin from the United States. Minority groups included African Americans, American Indians, Alaskan Natives, Asians and Pacific Islanders, Hispanics, and other ethnicities. Never-smokers were defined as those persons who had smoked < 100 cigarettes during their lifetime. Data collected from ever-smokers (former and current) included age of smoking initiation, years of smoking (duration), cigarettes smoked per day (intensity), and date of smoking cessation. A detailed environmental tobacco smoke history was also obtained.14

PI1 Allele Typing
To identify PI1 alleles, an isoelectric focusing (IEF) test (ie, {alpha}1-antitrypsin allele typing or phenotyping1516) was performed. IEF has been the standard clinical diagnostic test for A1ATD for > 20 years in the United States and Europe, and the proteins that have been separated in the electrophoresis gel are visualized with a Coomassie Blue protein stain.17 Over 70 variants of {alpha}1-antitrypsin have been reported, each named by a letter of the alphabet, are transmitted as a codominant gene (PI1).18 Over 60 of these variants are rare (ie, < 0.001 of allelic frequency) and are infrequently observed in the general population. The majority of the population is MM type or some combinations of its subtypes (ie, M1, M2, or M3), all of whom have normal serum {alpha}1-antitrypsin levels of 110 to 200 mg/dL.17 A1ATD (ie, serum {alpha}1-antitrypsin level, ≤ 80 mg/dL) is mainly seen in individuals with ZZ, SZ, SS, II, and null types.19 The serum {alpha}1-antitrypsin levels are marginally normal in those who persons who are heterozygous for the Z or S allele (serum {alpha}1-antitrypsin level, 70 to 110 mg/dL).19 The two common variants that produce the A1ATD phenotype are Z and S, and the rare variants include I, null, and others.9 We included all deficient alleles in the definition of PI1 allele type or high-risk allele type.

Quality control of the test results was exercised by the following four approaches: (1) one reference control sample was placed for every four lanes on each gel; (2) rare alleles was repeated next to the reference sample; (3) a quantitative assay was repeated to verify low values of < 100 mg/dL; and (4) when the allele type did not correspond to the {alpha}1-antitrypsin levels, both assays were repeated to rule out the possibility of mismatched samples. As a systematic measure of the error rate, IEF and nephelometry were repeated on 5% of the study subjects, and the results showed a 100% agreement between the original test and the repeated test.

Polymorphic Markers for Neutrophil Elastase Gene
ELA2 is the designated name (symbol) for the neutrophil elastase gene. ELA2 maps to chromosome 19p13·3 and is approximately 50 kb.2021 A workstation (NanoChip Molecular Biology workstation; Nanogen; San Diego, CA) was employed in determining the genotypes at two ELA2 promoter region single-nucleotide polymorphism (SNP) sites, –903 (Rep_a) and –741 (Rep_b).1022 Patient genomic DNA samples (20 ng) were amplified in a 384-well thermocycler (model 9700; ABI) in 20-µL reactions containing 0.5 µmol/L biotinylated primers (5' AGG ACC AGA GAA GTG CCT ATT GC 3'-FORWARD; 5' CAA ACC TGC CAA ACC TAG ACC TG 3'-REVERSE), 2 mmol/L MgCl2, and standard amounts of the remaining reagents. Forty 30-s cycles of polymerase chain reaction were performed at an annealing temperature of 58°C. The reactions were purified using polymerase chain reaction clean-up plates (MultiScreen; Millipore; Billerica, MA) and were rehydrated in 20 µL ddH2O. The average concentrations were approximately 10 ng/µL. Ten microliters of each sample was added to 35 µL 100 mmol/L histidine, which was brought to 70 µL with the addition of water, and was loaded onto a 96-pad microarray (NanoChips; Nanogen), which were subsequently probed with allele-specific 5' Cy dye-labeled oligos (ELA2–903 5'-cy5-GGC CCT GTG A, 5'-cy3- GGC CCT GTG C, stabilizer 5'-TAC CGG CCA CAT GCA GCT GTG TCG CC; ELA2–741 5'-cy5- CGG TAT CAC G, 5'-cy3-CGG TAT CAC G, and stabilizer 5' GGG CCC TGG GTA AAC TGA GGC A), and were scanned. The –903 T/G SNP was discriminated at 39°C in a 50 mmol/L sodium phosphate buffer and was easily genotyped based on dye signals. The –741G/A SNP was discriminated by a melting profile of 24 to 42°C. Quality control was exercised by the inclusion of genomic DNA control subjects in every plate. The use of a robotic workstation to aliquot DNA for each assay minimized sample switching. All data were reviewed by the laboratory director (J.M.C.) before analysis.

ELA2 Rep_a and Rep_b Haplotype Analysis
Haplotypes are specific combinations of nucleotides on the same chromosome. SNP markers within the same gene may not be independent, such as the two SNP sites in the ELA2 locus. They may be coinherited because of linkage disequilibrium (LD), and they may jointly influence the function of the resulting protein. We employed a new method to test the statistical association between haplotypes and phenotypes for case-control studies, as described by Schaid et al23 This method uses an expectation-maximization algorithm to infer haplotypes, and accounts for ambiguity in haplotype assignment when comparing case patients to control subjects and allows adjustment for nongenetic covariates (eg, tobacco smoking history), which are critical when analyzing genetically complex phenotypes like lung cancer. This method also provides several different global tests for association, as well as haplotype-specific tests, which give a meaningful advantage in attempting to understand the roles of many different haplotypes. We used D' and r2 to quantify and compare LD. Values of D' near unity and r2 of more than one third are considered to be indicative of a strong LD and of correlation between the two markers, respectively.

Case-Control Analysis
Our goal was to identify and quantify the elevated lung cancer risk imposed by PI1 and ELA2 genotypes (Table 1 ) in concert with known environmental and host factors. Standard contingency table methods were used for testing whether the high-risk genotype or haplotype frequency is different in lung cancer patients than in control subjects. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated2425 to quantify any significant association using logistic regression models. These models used case/control status as an outcome variable, and high-risk genotype, existing chronic lung disease, history of tobacco smoke exposure, and hypothesis-driven two-way interactions of these variables, as well as other known confounding variables (eg, family history), as possible predictor variables.2426 The magnitude of each effect was estimated by the variable-specific regression coefficient. The impact of family history on these models was adjusted by incorporating binary dummy variables into the conditional logistic regressions. Then multivariable models for genotypes and haplotypes were built to generate a lung cancer risk-predicting model system.2728 We obtained estimates of ORs and CIs for the inferred haplotypes using weighted logistic regression analysis (HaploStats program).23 Separate models were built under the alternative mendelian inheritance predictions of dominance between the inferred haplotypes of each person (ie, autosomal-dominant, additive [codominant], or recessive).232930 The best-fit model was judged by the information criteria of Akaike.31


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  		Table 1. Hypothetical {alpha}1-Antitrypsin and Neutrophil Elastase Imbalance and Predicted Effect on Lung Cancer Risk*

 

   Results
TOP
 Abstract
 Introduction
 Study Subjects and Methods
 Results
Discussion
 References
 
Control subjects were 2 years older than case patients, on average, but had a similar gender ratio and ethnic background (Table 2 ). Compared to case patients, control subjects were less likely to be cigarette smokers, to have a history of COPD or environmental tobacco smoke exposure, or to have a positive family history of lung cancer in their first-degree relatives.


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  		Table 2. Selected Characteristics of 305 Case Patients and 338 Control Subjects Enrolled in 1997–2002, Mayo Clinic*

 
Deficient PI1 types were significantly overrepresented among case patients compare to control subjects (p < 0.05) [Table 3 ]. For the two ELA2 SNP sites, the frequency distribution of alleles and genotypes at Rep_a was similar between case patients and control subjects, but differed at Rep_b. Specifically, the allele G at Rep_b was overrepresented among case patients. We then compared intragenic haplotypes between case patients and control subjects (Table 3, lower portion) using the haplotype score test.23 There was an overall strong association (judged by global and simulated p values) between the two SNPs and lung cancer risk, particularly with haplotypes T-G and G-A. These two SNPs showed a strong LD in the case patients (D' approximately equal to 1.0) and a weak LD in the control group (D' = 0.81), suggesting that they may have different functionality toward the development of lung cancer. No correlation existed between the two SNPs in case patients or control subjects (r2 = 0.01), reflecting the disparate allele frequencies of the two markers in the study population.28


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  		Table 3. Allele Frequencies and Haplotype Analysis of PI1 and ELA2 Genes

 
Since only a minority of our study subjects were non-US white persons, we repeated the analyses restricting them to US white persons only. The results for the entire study group and for US whites only were very similar. We provide information in Table 4 with regard to the frequencies of the PI1 allele type and the ELA2 haplotype among American Indians and other minority groups compared to the US whites in our study.


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  		Table 4. PI1 Allele and ELA2 Haplotype Frequencies by Ethnicity

 
To assess the effects of A1ATD allele types and Rep_a/Rep_b haplotypes on lung cancer risk, we built multivariable models estimating the independent and interactive effects of the two genes on lung cancer risk. All models were adjusted for age and gender, with tobacco smoke exposure, family lung cancer history, and COPD as covariates. Among the three alternative mendelian patterns, the dominant model best fitted the data, showing that carriers of the A1ATD allele or ELA2 haplotype T-G have a 2.0-fold or 4.1-fold increased risk, respectively (Table 5 ). Therefore, an individual with an {alpha}1-antitrypsin-deficient allele and a T-G haplotype had a 6.1-fold (mean [± SD], 2.0 + 4.1) increased risk of lung cancer. Under the same model, we observed the following significant interactive effect of the PI1 allele type and the ELA2 haplotype: carrying a haplotype G-G was not associated with an increased risk, but carrying both an {alpha}1-antitrypsin-deficient allele and a haplotype G-G carried a 2.6-fold higher risk for developing lung cancer (Table 5, lower portion).


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  		Table 5. Age and Gender-Adjusted Effects of A1ATD Allele and ELA2 Haplotypes on Lung Cancer Risk*

 

   Discussion
TOP
 Abstract
 Introduction
 Study Subjects and Methods
 Results
 Discussion
References
 
In our case-control study, we have found positive associations with genotypes indicating that an excess of neutrophil elastase or an insufficiency of {alpha}1-antitrypsin increases lung cancer risk. Our findings suggest that carrying a PI1-deficient allele (for a lower level of {alpha}1-antitrypsin) or a T-G haplotype (for a higher level of neutrophil elastase) is a risk factor for lung cancer. We did not observe an additionally elevated lung cancer risk for carrying both of these high-risk genotypes, indicating that they act independently. Our results are biologically plausible and support the hypothesis that an imbalance of {alpha}1-antitrypsin and neutrophil elastase, tobacco smoking, and COPD are significant risk factors for lung cancer (Fig 1 ). These three components, representing an inherited predisposition, exposure to environmental carcinogens, and pathologic host lung conditions, should be simultaneously considered in future investigations of lung cancer risk.



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  		Figure 1. Tobacco smoke, neutrophil elastase, {alpha}1-antitrypsin, chronic lung disease, and lung cancer.

 
{alpha}1-Antitrypsin is a serine protease inhibitor that binds and inhibits neutrophil elastase in the lung.32 Neutrophil elastase, a serine hydrolytic protease secreted primarily by neutrophils (ie, polymorphonuclear leukocytes) functions mainly to defend the host by degrading foreign microorganisms or organic molecules that have undergone phagocytosis by neutrophils.33 However, neutrophil elastase can also destroy the fundamental elastin-rich structure of lung tissue. Therefore, when the level of {alpha}1-antitrypsin is insufficient in the lung, neutrophil elastase destroys the elastin walls, the terminal respiratory unit (acini)171932 and has a critical role in the pathway between COPD and lung cancer risk.9

Only in the past few years have ELA2 mutations in the coding region been linked to two rare diseases, cyclic hematopoiesis (or cyclic neutropenia) and severe congenital neutropenia with unknown mechanisms.34353637 One study38 indicated that mutant neutrophil elastase may interfere with normal activity. Up-to-date searching for SNPs associated with the ELA2 gene in the National Center for Biotechnology Information dbSNP database (http://www.ncbi.nlm.nih.gov/SNP/) and the SNP Consortium (http://snp.cshl.org/) returned 22 SNPs. Eight SNPs are located in introns, and 14 are found in the upstream or downstream flanking regions, but no functional impact or population frequency information was available for any of these. Our research team reported three polymorphisms: T or G SNP at –903 base pairs (bp), A or G at –741 bp from the transcription initiation site, and an extra 52-bp repetitive sequence between the fourth and fifth repetitive motifs.10 The allele type –903TT or –741GG has been reported10 to be associated with increased lung cancer development. It is also worth noting that the 5' flanking region of the gene contains six tandem repeats of a 53-bp nucleotide sequence (REP53), which contains a potential binding site for a basic helix-loop-helix protein located at –1032 to –716.39 This sequence has been shown to have an enhancer function in transfection experiments, but the physiologic role of REP53 in the regulation of ELA2 activity in vivo has not been demonstrated.39

Years of research on A1ATD have given us a clear understanding of PI1 variations, functional significance, and association with disease status. However, knowledge of the ELA2 gene function is very limited. The functional significance of the Rep_a and Rep_b was examined by the luciferase activity of the promoter region containing different SNPs, which demonstrated a 1.9-fold higher relative luciferase activity in the promoter construct with –903T/-741G (T-G) compared to the –903G/-741A (G-A).10 These results provided evidence that the TT-GG type (or T-G haplotype) correlates with a high neutrophil elastase level. Individuals with this genotype might have an imbalance of {alpha}1-antitrypsin and neutrophil elastase, mimicking A1ATD.

An issue related to the study population is that a large number of new lung cancer case patients could be rapidly enrolled because Mayo Clinic is a major tertiary referral center. However, using self-referred or physician-referred patients as the case group raises the possibility of selection bias. For example, the ethnic backgrounds of all participants were predominantly whites from the United States, so the generalizability of the study might be compromised by the limited sample of underrepresented minorities.

An additional concern is the choice of the control group. Selecting control subjects in a tertiary referral clinic can be difficult. Since our lung cancer patients are primarily referred, one might think that the ideal control subjects should be matched with case patients by age, gender, race, geographic referral area, and duration of care at Mayo Clinic (ie, equally referred). However, this generates more concerns since these patients were typically referred for other disorders that may have been related to the conditions under study. In addition, approximately 60% of our lung cancer case patients were from outside of the tristate area (ie, Minnesota, Wisconsin, or Iowa) where a limited number of eligible control subjects can be found and enrolled. Therefore, we have chosen to use community residents as control subjects based on the principle of control selection by Mantel and Haenszel40 (ie, using a group representing a more general population could be superior if the comparability of the exposure is the major concern). Because the main exposures in our study are genetic traits, biases of preferential recall or exposure time are no longer major concerns. Moreover, our control group cannot only serve as a reference in testing our hypotheses but can also provide allele frequencies of the candidate markers in a defined population.

Despite these potential limitations, to our knowledge, our study is among the first published reports to specifically test the novel hypothesis that an imbalance of {alpha}1-antitrypsin and neutrophil elastase is important in lung cancer development. In addition, we addressed the risk-modifying role of PI1 and ELA2 genotypes in conjunction with tobacco exposure and host pathologic lung disease (eg, COPD). These results provide new insights with which to better understand the mechanistic basis of carcinogenesis and suggest directions for further research. Our results may also be useful in identifying potential feasible biological markers to assist lung cancer screening and early detection in order to provide opportunities for lung cancer risk reduction among former smokers and people with existing lung diseases.


   Acknowledgements
 
We would like to acknowledge James R. Jett, Jerry A. Katzmann, Noralane M. Lindor, Paul D. Scanlon, Stephen N. Thibodeau, and Victor F. Trastek for their contributions at various stages of this work. We also thank Susan Ernst for her technical assistance with the manuscript.


   Footnotes
 
This research was supported by grants NIH-CA 77118, NIH-CA 80127, and NIH-CA 84354 from the National Institutes of Health.

Abbreviations: A1ATD = {alpha}1-antitrypsin deficiency; bp = base pairs; CI = confidence interval; ELA2 = neutrophil elastase-2; IEF = isoelectric focusing; LD = linkage disequilibrium; OR = odds ratio; PI = protease inhibitor; SNP = single-nucleotide polymorphism

Received for publication December 7, 2004. Accepted for publication January 4, 2005.


   References
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 Abstract
 Introduction
 Study Subjects and Methods
 Results
 Discussion
 References
 

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