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Alpha-1 Antitrypsin Deficiency and Lung Cancer Risk

A Case-Control Study in Never-Smokers

      Background

      Never-smokers comprise up to 25% of all lung cancer cases. They could have different molecular pathways for lung cancer induction compared with smokers. Alpha-1 antitrypsin (AAT) deficiency is a hereditary trait whose main characteristic is early onset of lung emphysema. Our aim is to know if AAT-deficient carriers have a higher risk of lung cancer in a study performed exclusively in never-smokers.

      Methods

      We designed a multicentre hospital-based case–control study, which included incident never-smoking lung cancer cases. Controls were never-smokers attending nonmajor surgery at the participating hospitals. Controls were frequency matched on age and gender with cases. We determined AAT variants (alleles S and Z) through polymerase chain reaction.

      Results

      Two hundred and twelve cases and 318 controls were included. PiSS individuals showed a lung cancer risk of 4.64 (95% confidence interval: 1.08–19.92) compared with those with normal genotype (PiMM). When the analysis was restricted to women, the risk for PiSS increased to 7.58 (95% confidence interval: 1.40–40.87). This risk for homozygous SS was even higher for individuals exposed to environmental tobacco smoke (greater than 20 years). The presence of other alleles did not show any effect on lung cancer risk.

      Conclusions

      Never smoking SS homozygous individuals pose an increased risk of lung cancer. The risk is higher for individuals exposed to environmental tobacco smoke.

      Key Words

      Lung cancer is a worldwide health problem. It is currently the leading cause of cancer death.
      • Malvezzi M
      • Bertuccio P
      • Levi F
      • La Vecchia C
      • Negri E
      European cancer mortality predictions for the year 2013.
      Although the main risk factor is tobacco consumption, up to 25% of all cases are diagnosed in never-smokers, with important geographic variations.
      • Ferlay J
      • Shin HR
      • Bray F
      • Forman D
      • Mathers C
      • Parkin DM
      Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008.
      The most important risk factor in never-smokers is residential radon.
      • Torres-Durán M
      • Barros-Dios JM
      • Fernández-Villar A
      • Ruano-Ravina A
      Residential radon and lung cancer in never smokers. A systematic review.
      • Torres-Durán M
      • Ruano-Ravina A
      • Parente-Lamelas I
      • et al.
      Lung cancer in never smokers. A case–control study in a radon prone area (Galicia, Spain).
      Lung cancer in never-smokers has been proposed as a different disease than lung cancer occurring in ever-smokers due to different molecular pathways.
      • Sun S
      • Schiller JH
      • Gazdar AF
      Lung cancer in never smokers–a different disease.
      • Rudin CM
      • Avila-Tang E
      • Harris CC
      • et al.
      Lung cancer in never smokers: Molecular profiles and therapeutic implications.
      Nevertheless, the available studies are still scarce.
      Alpha-1 antitrypsin deficiency (AATD) is a hereditary condition first described by Laurell and Eriksson.
      • Laurell CB
      • Eriksson S
      The electrophoretic alfa1-globulin pattern of serum in alfa1-antitrypsin deficiency.
      Alpha-1 antitrypsin (AAT) is a glycoprotein codified by SERPINE1 gen, placed in the long arm of chromosome 14. It is synthesized mainly in the liver and its main function is to inhibit neutrophil elasthase and other serine proteases. It provides more than 90% of the total antiprotease capacity of the organism
      • Carrell RW
      • Lomas DA
      • Sidhar S
      • Foreman R
      Alpha 1-antitrypsin deficiency. A conformational disease.
      and, in the past years, it has been described that it has anti-inflammatory
      • Pott GB
      • Chan ED
      • Dinarello CA
      • Shapiro L
      Alpha-1-antitrypsin is an endogenous inhibitor of proinflammatory cytokine production in whole blood.
      and inmunomodulatory
      • Subramaniyam D
      • Virtala R
      • Pawłowski K
      • et al.
      TNF-alpha-induced self expression in human lung endothelial cells is inhibited by native and oxidized alpha1-antitrypsin.
      properties, among others.
      AAT gene comprises two alleles that are transmitted through a codominant autosomic Mendelian pattern. Wild alleles are named M and are present in 85%–90% of individuals (MM). The most frequent deficient alleles are called S and Z and are present in 10% and 2% of Spanish population, respectively,
      • Blanco I
      • Fernández-Bustillo E
      • de Serres FJ
      • Alkassam D
      • Rodríguez Menéndez C
      PI*S and PI*Z alpha 1-antitrypsin deficiency: Estimated prevalence and number of deficient subjects in Spain.
      although there are very few published studies. The severity of the deficiency is related to the deficient allele. The S allele expresses around 40% of AAT and the Z allele expresses around 15%.
      The main AATD clinical symptom is the early development of lung emphysema, mainly in individuals exposed to tobacco smoke. It is also known its association with bronchiectasis, panniculitis, and ANCA+ vasculitis. Some investigations have been published in the past years associating AATD with fibromyalgia or asthma.
      • Blanco LE
      • de Serres FJ
      • Fernández-Bustillo E
      • et al.
      Alpha1-antitrypsin and fibromyalgia: New data in favour of the inflammatory hypothesis of fibromyalgia.
      • Blanco I
      • Canto H
      • Flóres J
      • et al.
      Long-term augmentation therapy with alpha-1 antitrypsin in an MZ-AAT severe persistent asthma.
      • Blanco I
      • Lara B
      • de Serres F
      Efficacy of alpha1-antitrypsin augmentation therapy in conditions other than pulmonary emphysema.
      The imbalance protease–antiprotease originated from the low AAT concentration in blood and tissues causes a lower protection against proteases, being the most important neutrophil elastase, and is the cause of the harm that finally produces lung emphysema secondary to AATD. These alterations could favor the hypothesis of lung carcinogenesis.
      Some investigations have assessed the possible relation between AATD and the risk of lung cancer, with opposite results.
      • El-Akawi ZJ
      • Nusier MK
      • Zoughool FE
      Relationship between alpha-1 antitrypsin deficient genotypes S and Z and lung cancer in Jordanian lung cancer patients.
      • Topic AS
      • Jelic-Ivanovic ZD
      • Spasojevic-Kalimanovska VV
      • Spasic SM
      Association of moderate alpha-1 antitrypsin deficiency with lung cancer in the Serbian population.
      • Yang P
      • Wentzlaff KA
      • Katzmann JA
      • et al.
      Alpha1-antitrypsin deficiency allele carriers among lung cancer patients.
      • Yang P
      • Sun Z
      • Krowka MJ
      • et al.
      Alpha1-antitrypsin deficiency carriers, tobacco smoke, chronic obstructive pulmonary disease, and lung cancer risk.
      All these studies have included a high percentage of smokers and exsmokers, and there is no study performed exclusively in never-smokers. A study performed in never-smokers would eliminate the possible distortion caused by tobacco consumption and therefore increase the validity of the study results. This advantage would be greater should the study retrieved information on residential radon and environmental tobacco smoke.
      The aim of this study is to analyze if it exists an association between deficient AAT alleles, carried in homozygosis or heterozygosis and the risk of lung cancer in never-smokers. As a secondary objective, we aim to know if AATD could pose more risk in individuals exposed to environmental tobacco smoke for more than 20 years versus those exposed during a shorter period.

      PATIENTS AND METHODS

      We designed a multicentre hospital-based case–control study. Seven Galician hospitals and one in Asturias took part. Cases were all never-smoking lung cancer cases diagnosed at the participating hospitals between January 2011 and December 2013. Controls were never-smoking individuals who undergone nononcologic surgery, mainly major ambulatory surgery. To be classified as a never-smoker, we used the definition of the World Health Organization: to have smoked less than 100 cigarettes in lifetime or having smoked less than 1 cig/day during 6 months. Controls were frequency-matched with cases on age and gender.
      All participants were interviewed on their lifestyle, with special emphasis on environmental tobacco smoke exposure, previous occupations, leisure time activities, and diet and alcohol consumption. We measured residential radon in most participants’ homes after giving them a radon detector. The device was placed in the main bedroom at least during 3 months. Environmental tobacco smoke exposure was defined as having lived with a smoker at least during the past 20 years. Exposed individuals were asked about the relationship with the smoker/s at the same dwelling, years of living, and number of daily cigarettes smoked by the cohabitant.
      We searched for significant airflow obstruction in clinical records of all patients with AATD (homozygous SS or ZZ, and heterozygous MS, MZ, or SZ) and also searched for emphysema using lung computed tomography images. This information allowed us to rule out COPD or emphysema in these AATD lung cancer patients.
      Participants also donated 3 ml of total blood that was used to determine certain genetic polymorphisms, including deficient alleles for AAT S and Z, through analyzing the genotype. All samples were analyzed at the National Genotyping Center at the University of Santiago de Compostela. This facility has the most advanced techniques combined with rigorous quality control procedures.
      Genotyping was performed using the MassARRAY iPLEX GOLD SNP genotyping system (Sequenom Inc., San Diego, CA), following the manufacturer's instructions. The principles of this method are detailed in Buetow et al.
      • Buetow KH
      • Edmonson M
      • MacDonald R
      • et al.
      High-throughput development and characterization of a genomewide collection of gene-based single nucleotide polymorphism markers by chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
      The study protocol was approved by the Galician Ethics Committee (2010/295) and all participants signed a written consent to participate in this research.

      Statistical Analysis

      We first performed a univariate and bivariate analysis, describing the characteristics of cases and controls regarding age, gender, exposure to environmental tobacco smoke, and residential radon. Afterwards, we performed a logistic regression where the dependent variable was the case or control status and the main independent variable being a carrier of S or Z alleles in homozygosis or heterozygosis. We did the same analysis for women (but not for men due to the low number on included males) and finally we analyzed if the risk of lung cancer for the deficient AAT alleles was different depending on having being exposed or not to environmental tobacco smoke during the past 20 years. The results are expressed as ORs with 95% confidence intervals. The analysis was performed with SPSS version 20.

      RESULTS

      Two hundred and twelve cases and 318 controls were included. Table 1 shows the sample characteristics.
      TABLE 1Sample Description
      VariableCases, n (%)Controls, n (%)
      n = 212n = 318
      Age
       Median7070
       P25–P7561–7763.5–79
       Range34–8743–90
      Gender
       Female172 (81.1)250 (78.7)
       Male40 (18.9)68 (21.3)
      Education
       No formal studies54 (26.1)51 (16.0)
       Primary school124 (59.9)236 (74.2)
       High school15 (7.2)19 (6.0)
       University degree14 (6.8)12 (3.8)
      Residential radon exposure (Bq/m3)
       <20091 (52)188 (69.6)
       >20084 (48)82 (30.4)
      Exposure to ETS at home in past 20 years
       Yes95 (44.8)144 (45.4)
       No117 (55.2)173 (54.6)
      Histological types
       Adenocarcinoma164 (77.7)
       Squamous cell carcinoma20 (9.5)
       Small-cell carcinoma12 (5.7)
       Large-cell carcinoma7 (3.3)
       Other histological types8 (3.8)
      AAT mutations
       S
        Not present157 (74.1)220 (69.2)
        Heterozygosis48 (22.6)95 (29.9)
        Homozygosis7 (3.3)3 (0.9)
       Z
        Not present201 (94.8)302 (95)
        Heterozygosis11 (5.2)16 (5)
        Homozygosis00
      ETS, environmental tobacco smoke; AAT, alpha-1 antitrypsin.
      The median age of cases and controls was similar and also gender distribution, 81.8% of cases were women versus 78.7% of controls. Residential radon exposure was available for 175 cases (82.5%) and 270 controls (84.9%). Of them, 48% of cases were exposed to radon concentrations higher than 200 Bq/m
      • Torres-Durán M
      • Barros-Dios JM
      • Fernández-Villar A
      • Ruano-Ravina A
      Residential radon and lung cancer in never smokers. A systematic review.
      compared with 30.4% of controls. The predominant histological type was adenocarcinoma (77.7%) followed by squamous cell carcinoma (9.5%). We included 63 AAT-deficient cases. All these cases except one had a lung CT scan. We did not find any case of lung emphysema and only in three cases localized bronchiectasis were described. Lung function tests were available in 26 of 63 AATD cases. In those cases with spirometry, it was normal in 19, it was obstructive in three cases and it showed a nonobstructive pattern in four cases. These three patients were, respectively, MS, MZ, and SS. 22.6% of cases had a heterozygous allele S compared with 29.9% of controls. Seven cases (3.3%) and three controls (0.9%) were homozygous SS. Heterozygous Z carriers were observed in 11 cases (5.2%) and 16 controls (5%). There were no ZZ homozygous individuals. The distribution of the different combinations of alleles and their risk of lung cancer is shown in Table 2.
      TABLE 2AAT Genotype and Risk of Lung Cancer
      Cases, n (%)Controls, n (%)OR (95% CI)
      OR adjusted by age and gender.
      pOR (95%CI)
      OR adjusted by age, gender, ETS, and residential radon exposure (<200, ≥200 Bq/m3).
      p
      AAT
       MM149 (70)208 (65.4)1 (−)1 (−)
       MS45 (21.1)91 (28.6)0.66 (0.41–1.05)0.080.67 (0.42–1.08)0.10
       MZ8 (3.8)12 (3.8)0.75 (0.27–21.09)0.580.85 (0.30–2.40)0.76
       SZ3 (1.4)4 (1.3)1.25 (0.27–5.78)0.780.94 (0.20–4.48)0.94
       SS7 (3.3)3 (0.9)3.38 (0.81–13.99)0.094.64 (1.08–19.92)0.04
      ETS exposure
       No117 (55.2)173 (54.6)1 (−)
       Yes95 (44.8)144 (45.4)0.78 (0.52–1.18)0.25
      Radon exposure
       <200 Bq/m
      • Torres-Durán M
      • Barros-Dios JM
      • Fernández-Villar A
      • Ruano-Ravina A
      Residential radon and lung cancer in never smokers. A systematic review.
      91 (52)188 (69.6)1 (−)
       ≥200 Bq/m
      • Torres-Durán M
      • Barros-Dios JM
      • Fernández-Villar A
      • Ruano-Ravina A
      Residential radon and lung cancer in never smokers. A systematic review.
      84 (48)82 (30.4)2.36 (1.60–3.58)<0.01
      AAT, alpha-1 antitrypsin; OR, odds ratio; CI, confidence interval; ETS, environmental tobacco smoke.
      a OR adjusted by age and gender.
      b OR adjusted by age, gender, ETS, and residential radon exposure (<200, ≥200 Bq/m3).
      It can be observed that there is no apparent risk of lung cancer for heterozygous MS or MZ and neither for SZ patients. On the opposite, the adjusted risk for homozygous SS individuals is 4.64 (95% confidence interval [CI]: 1.08–19.92). When the analysis is restricted to women (Table 3), there is no significant effect for the different combinations of heterozygous MS, MZ, or SZ. For heterozygous SS women the risk of lung cancer is 7.58 (95% CI: 1.40–40.87).
      TABLE 3AAT Genotype and Lung Cancer Risk in Never-Smoking Women
      Cases, n (%)Controls, n (%)OR (95% CI)
      OR adjusted by age.
      pOR (95% CI)
      OR adjusted by age, ETS, and residential radon exposure (<200, >200 Bq/m3).
      p
      AAT
       MM120 (69.8)162 (64.8)1 (−)1 (−)
       MS34 (19.8)73 (29.2)0.68 (0.40–1.16)0.160.70 (0.41–1.21)0.21
       MZ8 (4.6)11 (4.4)0.86 (0.30–2.44)0.771.06 (0.36–3.07)0.92
       SZ3 (1.7)2 (0.8)2.44 (0.39–15.15)0.342.02 (0.31–13.01)0.46
       SS7 (4.1)2 (0.8)5.02 (0.97–25.83)0.057.58 (1.40–40.87)0.02
      ETS exposure
       No85 (49.4)121 (48.6)(−)1 (−)
       Yes87 (50.6)128 (51.4)(−)0.77 (0.49–1.21)0.25
      Radon exposure
       <200 Bq/m
      • Torres-Durán M
      • Barros-Dios JM
      • Fernández-Villar A
      • Ruano-Ravina A
      Residential radon and lung cancer in never smokers. A systematic review.
      85 (54.5)155 (73.1)(−)1 (−)
       ≥200 Bq/m
      • Torres-Durán M
      • Barros-Dios JM
      • Fernández-Villar A
      • Ruano-Ravina A
      Residential radon and lung cancer in never smokers. A systematic review.
      71 (45.5)57 (26.9)(−)2.80 (1.74–4.51)<0.01
      AAT, alpha-1 antitrypsin; OR, odds ratio; CI, confidence interval; ETS, environmental tobacco smoke.
      a OR adjusted by age.
      b OR adjusted by age, ETS, and residential radon exposure (<200, >200 Bq/m3).
      Table 4 displays the results for the combination of the different AAT genotypes combined with the exposure to environmental tobacco smoke (measured as having lived with a smoker during the past 20 years). It can be observed that when the results are adjusted by age, gender, and residential radon exposure, homozygous SS individuals exposed to environmental tobacco smoke present a lung cancer risk of 12.10 (95% CI: 1.18–123.77), while those not exposed pose an OR of 1.75 (95% CI: 0.23–13.11), although there were only two SS homozygous cases in this last category. It was not possible to analyze the relation between SS genotype and the risk of lung cancer for the different histological types because all SS lung cancer cases were adenocarcinomas. This information might suggest specificity for AATD for this histological type.
      TABLE 4AAT Genotype and Lung Cancer Risk by Environmental Tobacco Smoke Exposure
      Not exposed to ETSExposed to ETS
      Cases, n (%)Controls, n (%)OR (95% CI)
      OR adjusted by age and gender.
      POR (95% CI)
      OR adjusted by age, gender, and residential radon exposure.
      PCases, n (%)Controls, n (%)OR (95% CI)
      OR adjusted by age and gender.
      POR (95% CI)
      OR adjusted by age, gender, and residential radon exposure.
      P
      AAT
       MM81 (69.2)109 (63.0)1 (−)1 (−)68 (71.6)98 (68.1)1 (−)K-)
       MS28 (23.9)53 (30.6)0,63 (0.35–1.15)0.130.68 (0.37–1.25)0.2117 (17.9)38 (26.4)0.66 (0.31–1.41)0.280.57 (0.26–1.26)0.17
       MZ4 (3.4)6 (3.5)0.37 (0.07–1.93)0.240.39 (0.07–2.05)0.274 (4.2)6 (4.2)1.27 (0.32–4.97)0.731.65 (0.41–6.69)0.48
       SZ2 (1.7)3 (1.7)0.94 (0.15–6.02)0.950.84 (0.13–5.42)0.851 (1.1)1 (0.7)1.99 (0.12–33.88)0.631.11 (0.06–19.19)0.94
       SS2 (1.7)2 (1.2)1.42 (0.19–10.51)0.731.75 (0.23–13.11)0.585 (5.3)1 (0.7)7.32 (0.77–69.49)0.0812.10 (1.18–123.77)0.04
      Radon exposure
       <200 Bq/m
      • Torres-Durán M
      • Barros-Dios JM
      • Fernández-Villar A
      • Ruano-Ravina A
      Residential radon and lung cancer in never smokers. A systematic review.
      64 (40.4)107 (72.3)1 (−)41 (46.6)80 (66.1)K-)
       ≥200 Bq/m
      • Torres-Durán M
      • Barros-Dios JM
      • Fernández-Villar A
      • Ruano-Ravina A
      Residential radon and lung cancer in never smokers. A systematic review.
      42 (39.6)41 (27.7)1.75 (0.99–3.07)0.0547 (53.4)41 (33.9)3.48 (1.83–6.64)<0.01
      AAT, alpha-1 antitrypsin; OR, odds ratio; CI, confidence interval; ETS, environmental tobacco smoke.
      a OR adjusted by age and gender.
      b OR adjusted by age, gender, and residential radon exposure.

      DISCUSSION

      The results of this study show for the first time for never-smokers that SS homozygous individuals pose a fourfold lung cancer risk compared with those with the MM genotype. When the analysis is restricted to women the risk is seven times higher. We have also observed that the risk of lung cancer increases considerably for individuals with the SS genotype exposed to environmental tobacco smoke compared with those not exposed to environmental tobacco smoke. We have observed no effect for other AAT-deficient genotypes.
      A very relevant aspect of our study is having included only never-smokers because we avoid possible biases caused by tobacco consumption on the results’ interpretation. Individuals who are AAT deficient are more susceptible to environmental aggressions and tobacco is the main determinant of lung damage and it causes the appearance of lung emphysema and COPD.
      • Mayer AS
      • Stoller JK
      • Vedal S
      • et al.
      Risk factors for symptom onset in PI*Z alpha-1 antitrypsin deficiency.
      • Demeo DL
      • Sandhaus RA
      • Barker AF
      • et al.
      Determinants of airflow obstruction in severe alpha-1-antitrypsin deficiency.
      We reviewed lung CT scans and lung function tests in our AATD lung cancer cases. No cases of emphysema were described, and only in three cases localized, nonextensive, bronchiectasias were described. Not all lung cancer cases had a lung function test available in their medical chart, but among those cases whose lung function test could be reviewed we only found three with an obstructive pattern. In our opinion, these data show that there is no apparent association between emphysema or COPD with lung cancer in our sample of never-smokers. Furthermore, should an obstructive pattern be present, much more than half of our AATD patients would have a spirometry. This aspect supports the lack of association between AATD and an obstructive pattern in never-smoking lung cancer patients.
      The SS genotype implies a moderate AATD. The increase in the risk of lung cancer caused by environmental tobacco smoke for those SS homozygous could be explained because tobacco particles could attract neutrophils to lung alveoli in response. Neutrophils release proteases in the alveoli and the most important is neutrophil elastase, which has to be neutralized by AAT. In patients with AATD this antiprotease capacity is diminished which might increase the risk of lung damage.
      • Lomas DA
      • Parfrey H
      Alpha1-antitrypsin deficiency. 4: Molecular pathophysiology.
      AATD has two relevant characteristics. It is an infrequent genetic condition and this means that it is very difficult to perform studies with enough sample size to obtain results confirming or disregarding possible associations. It is also a very polymorphic gene and there exist ample variability with race and geography on the distribution of the different alleles. This fact makes difficult to generalize the results obtained by different studies.
      Numerous studies have been published on the frequencies of normal (M) and deficient alleles (S and Z) and some of them in Spanish population. A study performed in Asturias,
      Spain,
      • Blanco I
      • Fernández E
      • Rodríguez MC
      • Fernández A
      Allelic frequency of the gene of alpha-1-antitrypsin in the general population in a county in Asturias.
      observed frequencies of PiS and PiZ of 100 and 20 per 1000 inhabitants, respectively. Other study performed in Barcelona
      • Vidal R
      • Miravitlles M
      • Jardí R
      • et al.
      Study of the frequency of different phenotypes of alpha-1-antitrypsin in a population of Barcelona.
      including 440 participants observed frequencies of 104 and 16 per 1000. The first study performed in Spanish population was performed in Oslo in 1968 and it included 378 sailors, most of them from Galicia.
      • Fagerhol MK
      • Tenfjord OW
      Serum Pi types in some European, American, Asian and African populations.
      It found frequencies of PiS and PiZ of 112 and 12 per 1000. Our study shows that the frequencies of the deficient alleles S and Z are higher than those described previously in Spain. We have not detected homozygous ZZ and the distribution of the other genotypes is similar between cases and controls with the exception of homozygous SS, whose frequency is higher in cases than control (3.3% vs. 0.9%). It is known that there is a gradient Northwest-Southeast in Spain regarding deficient AAT individuals and this fact could justify the higher prevalence of deficient alleles that we have observed in Galicia.
      • Blanco I
      • Fernández E
      Alpha1-antitrypsin Pi phenotypes S and Z in Spain: An analysis of the published surveys.
      In a previous review published in 1998 on the frequencies of deficient alleles, the highest rates of S mutations were observed in Galicia and Portugal. The authors suggested that probably S mutation was originated in this geographical area in an unknown date.
      • Hutchison DC
      Alpha 1-antitrypsin deficiency in Europe: Geographical distribution of Pi types S and Z.
      We should also highlight that the available studies have not used the genotyping technology of our study and allele frequencies have been estimated previously using the phenotype,
      • Blanco I
      • Fernández E
      • Rodríguez MC
      • Fernández A
      Allelic frequency of the gene of alpha-1-antitrypsin in the general population in a county in Asturias.
      • Vidal R
      • Miravitlles M
      • Jardí R
      • et al.
      Study of the frequency of different phenotypes of alpha-1-antitrypsin in a population of Barcelona.
      • Andrés Puertas MJ
      • Arranz Peña ML
      Evaluation of alpha 1-antitrypsin phenotypes in Castilla-León: Study of a population of newborn infants.
      which is a less reliable method.
      Environmental tobacco smoke has been classified as a human carcinogen
      • IARC Working Group on the Evaluation of Carcinogenic Risks to Humans
      Tobbaco smoke and involuntary smoking.
      • Environmental Protection Agency
      and this study suggests a possible interaction between this exposure and AATD, but only for homozygous SS. Five of seven SS individuals have been exposed to environmental tobacco smoke for more than 20 years and lung cancer risk for them is 12.10 (1.18–123.77) while in those not exposed (n = 2) the risk is not statistically significant 1.75 (0.23–13.11).
      There are various carcinogenic mechanisms that have been proposed for lung cancer development in AATD individuals. These are mainly derived from the excess of neutrophil elastase, which could facilitate tissue damage and air trapping favoring a longer exposure to carcinogens in the alveoli. It can also promote tumor progression through the tumor necrosis factor receptor signaling pathway,
      • Sun Z
      • Yang P
      Role of imbalance between neutrophil elastase and alpha 1-antitrypsin in cancer development and progression.
      by inhibition of apoptosis. Neutrophil elastase can activate matrix metalloproteinases, a group of enzymes that have a role in tumor invasion and metastasis.
      • Shamamian P
      • Schwartz JD
      • Pocock BJ
      • et al.
      Activation of progelatinase A (MMP-2) by neutrophil elastase, cathepsin G, and proteinase-3: A role for inflammatory cells in tumor invasion and angiogenesis.
      Some studies have suggested that high levels of neutrophil elastase, main substrate of AAT, could have a role in lung carcinogenesis, not only in AATD but even in individuals with normal AAT concentration. Seric levels of neutrophil elastase are defined by ELA2 gen that have two known polymorphisms and they encode and produce a different amount of protein.
      • Yang P
      • Bamlet WR
      • Sun Z
      • et al.
      Alpha1-antitrypsin and neutrophil elastase imbalance and lung cancer risk.
      • Topic A
      • Ljujic M
      • Nikolic A
      • et al.
      Alpha-1-antitrypsin phenotypes and neutrophil elastase gene promoter polymorphisms in lung cancer.
      Other studies have observed that lung cancer risk increases in AATD individuals, but all of them included a high proportion of smokers and ex-smokers. Yang et al. observed a 13.4% of carriers of a deficient allele among cases and 7.8% among controls. In this study, being a carrier of one or two deficient alleles was associated with a lung cancer risk of 1.7 (95% CI: 1.2–2.4).
      • Yang P
      • Sun Z
      • Krowka MJ
      • et al.
      Alpha1-antitrypsin deficiency carriers, tobacco smoke, chronic obstructive pulmonary disease, and lung cancer risk.
      Other study performed in Serbian population
      • Topic AS
      • Jelic-Ivanovic ZD
      • Spasojevic-Kalimanovska VV
      • Spasic SM
      Association of moderate alpha-1 antitrypsin deficiency with lung cancer in the Serbian population.
      found a prevalence of deficient heterozygous alleles of 5.9% in cases and 3.7% in controls. Being a heterozygous-deficient carrier Z or S was associated with lung cancer risk (squamous cell histological type) of 4.51 (95% CI: 1.66–12.29), whereas no risk excess was observed for other histological types. We were not able to analyze other histological types because all homozygous SS were adenocarcinomas. This histological type comprised 77.7% of all cancer cases in our study.
      All previously published studies have included a wide majority of ever-smokers and a very low number of never-smokers that have not been analyzed separately. It is known that protease–antiprotease imbalance can increase lung tissue damage in response to exposure to tobacco smoke but in never smoking people the relevance of this pathway is unclear. In fact, there is a high proportion of never-smokers with severe AAT deficiency (PiZZ) that does not develop pulmonary emphysema in their lifetime. This information suggests that more studies are necessary to explore the possibility of an alternative pathway for lung carcinogenesis in never-smoking AAT-deficient individuals.
      This study has some limitations. The main one is the low number of deficient individuals (heterozygous and homozygous), but this limitation is common to all available studies. Other limitation is that we have not measured serum levels of AAT nor its main substrate, neutrophil elastase. Some authors have hypothesized that imbalance between AAT and neutrophil elastase may be critical in the causal pathway from tobacco smoke exposure to lung cancer development (33) but its specific role in the carcinogenesis in never-smokers is unknown. Very few studies have measured neutrophil elastase and this practice should be recommended in investigations evaluating AATD.
      Regarding seric levels of AAT, it is well defined in the literature that S allele implies a moderate AAT deficiency with a consequent reduction of antielastase activity. We did not measured AAT levels due to logistic reasons. Many studies assessing AAT role have not also measured AAT dosage in serum and it is important to have its levels to have a better picture of the effect of AATD. We could take as a reference for participants in our study the results of a previous study from Vidal et al done in Spanish population (24) where plasmatic levels of AAT associated with each genotype were: MM: 103–200 mg/dl, MS: 100–180 mg/dl, SS: 70–105 mg/dl, MZ: 66–120 mg/dl, SZ: 45–80 mg/dl, ZZ: 10–40 mg/dl. A last limitation is that we have not analyzed polymorphisms of other genes that might play a role in lung cancer onset.
      Our study has also important advantages. It has included only never-smokers, a relatively high number of cases and controls, we have measured environmental tobacco smoke exposure and also residential radon exposure. There are no published studies on AATD with these characteristics. The most important advantage is that we have been able to include a relatively high sample size comprised of never-smokers, allowing assessing the effect of AAT-deficient alleles S and Z without confounding by tobacco consumption.
      To conclude, the results of this study suggest that being a homozygous SS increases importantly the risk of lung cancer in never-smokers. This risk is higher for women and for individuals exposed to environmental tobacco smoke, suggesting a possible interaction. AATD is an under diagnosed condition and a potential deficiency should be considered for never-smoking lung cancer cases with adenocarcinoma.

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