1 Wayne State University School of Medicine, Detroit, MI, USA, 2 University of Michigan School of Public Health, Ann Arbor, MI, USA, 3 Molecular Biology and Human Genetics Program and 4 Population Studies and Prevention Program, Karmanos Cancer Institute at Wayne, State University, Detroit, MI, USA
* To whom correspondence should be addressed at: 110 E. Warren Avenue, Detroit, MI 48201, USA Email: cotem{at}med.wayne.edu
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Abstract |
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Abbreviations: PAHs, polycyclic aromatic hydrocarbons; GSTs, glutathione S-transferases; GSTM1, glutathione S-transferase µ; GSTP1, glutathione S-transferase ; GSTT1, glutathione S-transferase
; MDCSS, Metropolitan Detroit Cancer Surveillance System; SEER, Surveillance, Epidemiology and End Results
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Introduction |
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Susceptibility to tobacco smoke is likely to result from each individual's capacity to activate and detoxify carcinogens. Cytochrome P450 enzymes (i.e. Phase I enzymes) oxidize procarcinogens found in tobacco smoke, resulting in reactive metabolites that damage DNA. Phase II enzymes protect a cell by detoxifying these metabolites into compounds that are readily excreted from the body (9). Glutathione S-transferases (GSTs) are a class of Phase II enzymes that are present in many tissues, including lung (10). Alpha (), kappa (
), mu (µ), sigma (
), zeta (
), pi (
), theta (
) and omega (
) (11) are the eight major classes of genes known. Polymorphisms in glutathione S-transferase µ (GSTM1), glutathione S-transferase
(GSTT1) and glutathione S-transferase
(GSTP1) genes in humans are associated with a lack or a reduction of enzymatic activity toward several substrates, including those found in tobacco smoke (1215).
A common deletion polymorphism in the GSTM1 gene on chromosome 1p13.3 renders the enzyme inactive (16), thereby eliminating this route of conjugation and detoxification of reactive metabolites, particularly epoxide metabolites of PAHs (17). The GSTT1 gene, located on chromosome 22q11.23, has also been found to have a deletion polymorphism that results in an absence of enzyme activity (18). GSTT1 participates in detoxification of carcinogenic intermediates produced from tobacco smoke, namely monohalomethanes and butadiene. GSTP1 detoxifies PAHs and is the most abundant GST isoform in the lungs (10). A single nucleotide polymorphism in exon 5 (Ile105Val, 313A313G) of the GSTP1 gene, located on chromosome 11q13, results in significantly lower conjugating activity among individuals who carry one or more copies of the G (guanine) allele (Ile/Val or Val/Val) compared with those who have the A/A (adenine/adenine; Ile/Ile) genotype (19). Having at least one copy of the G allele in GSTP1 is also associated with increased levels of hydrophobic adducts in the lung and higher levels of PAHDNA adducts in human lymphocytes (20). Because of their role in the metabolism of carcinogens in tobacco smoke, these polymorphisms are potential modulators of lung cancer risk.
While risks associated with these polymorphisms have been studied in lung cancer, little attention has been paid to early-onset disease, where genetic susceptibility to lung carcinogens may be particularly important. To investigate the potential role of these GSTs, alone and in combination, as modulators of early-onset lung cancer risk, a population-based case-control study of early-onset lung cancer was performed.
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Materials and methods |
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This protocol was approved by the local institutional review board. Informed consent was obtained from all subjects prior to initiation of the interview and again prior to biospecimen collection. Trained interviewers conducted structured telephone interviews to collect demographic information, health history, cigarette use and family history of lung cancer in a first degree relative.
Biological samples with GST genotyping results were available for 350 interviewed cases and 410 interviewed controls. Owing to the rapidly fatal nature of lung cancer, it was necessary to collect 118 (33.7%) paraffin-embedded tumor blocks to obtain DNA from deceased cases. The use of these blocks as the DNA source for genotyping could lead to bias if the tumor tissue from the blocks contained deletions or other mutations not seen in normal tissue obtained from the blocks. When the genotype frequencies by tissue type (normal, tumor or mixed normal and tumor) were examined using Fisher's exact test, there were no differences in genotyping results by tissue type for GSTM1 (P-value = 0.79), GSTT1 (P-value = 0.40) or GSTP1 (P-value = 0.55). Any bias from the use of DNA from tumor blocks is minimal. The remainder of DNA samples in case subjects was extracted from either venous blood (44.6%) or buccal cells (21.7%). All DNA from control subjects were collected through blood (67.1%) or buccal cells (32.9%).
Genotyping
DNA was isolated from whole blood with the Genomic DNA Purification System (Gentra Systems, Minneapolis, MN), from buccal swabs with the BuccalAmpTM DNA Extraction Kit (Epicentre Technologies, Madison, WI) or from paraffin-embedded tissue with the QIAamp DNA Mini Kit (Qiagen, Valencia, CA) following the manufacturers' protocols. When multiple biospecimens were obtained from a study participant, DNA extracted from blood was used preferentially, followed by those from buccal swabs and then from normal tissue within paraffin blocks.
DNA isolated from buccal cells or paraffin embedded tissue was preamplified using a nested PCR strategy. Preamplification (outer amplification) was carried out in a 25 µl reaction mixture containing 2.5 mmol/l MgCl2, 0.5 µmol/l of the gene specific-primers (Table I)outer forward (OF) and reverse (OR), 1.25 U AmpliTaq Gold polymerase and 200 µmol/l of dATP, dCTP, dGTP and dTTP. The outer-amplification mixture was denatured at 95°C for 10 min and amplification was achieved by 15 cycles at 94°C for 30 s, 63°C for 30 s, and 72°C for 1 min, followed by a final extension at 72°C for 10 min. The outer amplification was performed on a Mastercycler® Gradient thermocycler (Eppendorf, Westbury, NY).
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The GSTM1 null mutation was assayed using quantitative PCR methods described earlier (21). Briefly, 25 ng of DNA extracted from whole blood or 2 µl of preamplification reaction mixture was mixed with forward (GSTM1-194F) and reverse (GSTM1-273R-2) primer and the product was detected with a fluorescent probe (GSTM1-215T) at an annealing temperature of 55°C.
The GSTT1 null mutation was determined as previously described (22). Briefly, following amplification with annealing temperature at 58°C, the PCR products were electrophoresed through 3% NuSieve 3:1 agarose (FMC) and visualized with ethidium bromide.
Primers for amplification of the region of the GSTP1 gene containing the functional polymorphism and fluorescent probes for allelic discrimination, were designed using Primer Express software (Applied Biosystems). Standard TaqMan conditions were applied in an ABI PRISM 7900 Sequence Detection System (Applied Biosystems) for amplification and detection of both alleles. Briefly, 25 ng of DNA derived from whole blood or 2 µl of preamplification reaction mixture, was added to 1x Universal PCR mix (Applied Biosystems) containing forward (GSTP1-IF) and reverse (GSTP1-IR) primers and GSTP1 A313 allele (GSTP1-A) and GSTP1 G313 allele (GSTP1-G) probes at an annealing temperature of 64°C.
For GSTM1 genotyping, there were four amplification failures among the controls and five among the cases. GSTT1, had 12 amplification failures among the controls and 19 among the cases. For GSTP1, there were three amplification failures among the controls and six among the cases. The remaining missing genotypes (25 for GSTM1, 25 for GSTT1 and 27 for GSTP1) were all in case subjects, when there was insufficient sample available to perform genotyping.
Statistical analysis
Distributions of demographic variables were compared between cases and controls using 2-tests for categorical variables and t-tests for continuous variables. Unconditional logistic regression models were used to estimate odds ratios (OR) and 95% confidence intervals (CI) to measure the association between lung cancer and genotype. All analyses presented in the Tables (II
V) were stratified by race. Potential confounders, including sex, age at diagnosis or interview (continuous), family history of lung cancer in a first degree relative (dichotomous) and pack years of cigarette smoking (continuous) were included in the final models. Subjects were categorized as ever smokers if they reported smoking at least 100 cigarettes in their lifetime. Pack years were calculated using the reported number of cigarettes smoked per day and the number of years smoked. Light smokers were those who smoked less than or equal to the mean number of pack years among smoking controls in each racial group (
18 pack years of smoking in Caucasians,
14 in African Americans); heavy smokers were those who smoked more than the mean number of pack years (
19 pack years of smoking in Caucasians,
15 in African Americans). GSTM1 null homozygotes, GSTT1 null homozygotes and GSTP1 A/G and G/G genotypes were considered to be risk genotypes for lung cancer development in this analysis. To evaluate the risk associated with combinations of genotypes, individuals were categorized as having 0, 1, 2 or 3 of the risk genotypes, with those in the 0 category used as the reference group.
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Results |
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Table III presents the distribution of genotypes by race and casecontrol status. A statistically significant difference by race in GSTM1 genotype distribution was identified in the control population, with 47.4% of Caucasian controls and 26.1% of African American controls being GSTM1 null (2 P-value < 0.0001). GSTT1 genotype frequency in controls did not differ by race (
2 P-value = 0.43), nor did the allele frequency for GSTP1 (
2 P-value = 0.19). GSTP1 genotype distribution in both Caucasian and African American controls was in HardyWeinberg equilibrium (
2 P-value = 0.47 and 0.36, respectively, data not shown).
Table IV reports the risk of early-onset lung cancer for each genotype in Caucasians, by smoking status. To investigate differences in genetic risk by smoking history, non-smokers were excluded and smokers were stratified into light or heavy smokers. Wenzlaff et al. (24) examined the risk of lung cancer in non-smokers associated with these genotypes. No increased risk by genotype in Caucasian light smokers was identified. In contrast, Caucasian heavy smokers showed a 3.1-fold increased risk of early-onset lung cancer in individuals who carried the GSTT1 null genotype compared with those who carried the GSTT1 functional genotype (95% CI 1.18.4) after adjusting for age, sex, pack years and family history of lung cancer (Table IV). Interactions between genotype and smoking exposure were also examined. In Caucasians, the interactions between pack years and GSTM1 (P = 0.02) and pack years, and GSTT1 (P = 0.02) were statistically significant, although no significant interaction between pack years and GSTP1 (P = 0.76) was identified.
The effects of combinations of risk genotypes on early-onset lung cancer risk were also examined. Caucasian heavy smokers with GSTM1 or GSTT1 null genotypes were 2.2-fold more likely to have early-onset lung cancer compared with those with GSTM1 and GSTT1 non-null, after adjusting for age, family history of lung cancer and pack years of smoking (95% CI 1.14.3). Risk increased 5-fold for heavy smokers with both GSTM1 and GSTT1 null genotypes (95% CI 1.123.6), and the test for trend revealed increased risk as the number of risk genotypes increased (P-value for trend = 0.005) (Table IV). Risk was also high for Caucasian heavy smokers who carried the GSTT1 null and GSTP1 risk genotypes compared with their counterparts with fully functional GSTT1 and GSTP1 genes (OR = 4.6 95% CI 1.022.1), although statistical significance was not attained.
Table V reports the risk of early-onset lung cancer for each genotype in African Americans, by smoking status. Analysis of single genotypes in African Americans suggested no significant associations between GSTM1 or GSTT1 null genotypes and early-onset lung cancer, after adjustment. However, African Americans carrying at least one G allele at the GSTP1 locus were 2.9-fold more likely to have lung cancer compared with African Americans without a G allele (95% CI 1.36.6). No significant interaction between pack years of smoking and GSTM1 (P = 0.15), GSTT1 (P = 0.62) or GSTP1 (P = 0.87) were identified.
The effects of the combinations of risk genotypes on early-onset lung cancer risk were also examined in African Americans. A statistically significant interaction between the GSTM1 and GSTT1 genotypes was observed (LRT 2 = 4.8, P-value = 0.03) (data not shown) with risk increased for carriers of both risk genotypes relative to a decreased risk in carriers of only one risk genotype. African Americans with either GSTT1 null or GSTP1 risk genotypes were 3.8-fold more likely to have lung cancer compared with African Americans with fully functioning GSTT1 and GSTP1 genes (95% CI 1.49.9). African Americans with either one or two risk genotypes at the GSTM1 and GSTP1 loci were at higher risk of having lung cancer compared with those with fully functional GSTM1 and GSTP1 genes (one risk genotype OR = 2.8, 95% CI 1.17.2 and two risk genotypes OR = 4.0, 95% CI 1.312.2; P-value trend for = 0.02). Stratification by smoking history in African Americans did not reveal any statistically significant differences between amount smoked and genotype, although the P-values for trend were close to significance in the light smoking category.
For all subjects combined, risk of early-onset lung cancer increased as the number of GST risk genotypes an individual carried increased (one risk genotype OR = 1.4, 95% CI 0.82.4, two risk genotypes OR = 1.7, 95 % CI 1.03.1 and 3 risk genotypes OR = 2.3 95% CI 1.05.4, P-value for trend = 0.03) after adjustment for age, race, sex, pack years of smoking and family history of lung cancer (data not shown). A significant trend for increasing risk with increasing number of GST risk genotypes was also seen in heavy smoking Caucasians (P-value for trend = 0.03).
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Discussion |
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Single gene effects
No statistically significant association was observed between GSTM1 null genotype and lung cancer in Caucasians or African Americans regardless of level of smoking in our early-onset population, with ORs of 1.3 and 1.5 in Caucasians and African Americans, respectively. A pooled analysis of Caucasian early-onset (age <45 years) lung cancer cases reported similar findings (25). These findings are also consistent with two meta-analyses that reported moderately (<20%) increased risk of lung cancer associated with the GSTM1 null genotype (27,28). The majority of the studies included in the meta-analyses, individually, did not find statistically significant increases in risk associated with GSTM1 genotype and lung cancer; however, taken together, significance was attained. Similar to the odds ratio reported here for African Americans (OR = 1.5), Ford et al. reported a 2.1-fold increase in the risk of lung cancer in African Americans associated with a GSTM1 null genotype after adjusting for age, sex and smoking (95% CI 1.14.1) (29). Given the current literature, GSTM1 genotype is a likely risk factor for lung cancer, albeit one with a small magnitude of effect. Risk may be most evident in certain ethnic populations, histological types or when analyzed in genegene and geneenvironment interactions (3033).
Heavy smoking Caucasians in our study population were found to be at a higher risk of early-onset lung cancer if they carried a null GSTT1 genotype. Hou et al. (34) reported a geneenvironment interaction for increased risk of lung cancer associated with GSTT1 null genotype in a small number of heavy smokers (>23 pack years) compared with light smokers with GSTT1 null genotypes (OR = 9.3 95% CI 1.946.3). Another study of 144 Caucasian early-onset cases (<45 years) found borderline increased risk, after adjusting for age, sex and study number (OR = 1.2, 95% CI 1.01.6) (25). Two other publications to date reported statistically significant increased risk of lung cancer associated with GSTT1 null genotype in Caucasians (35,36). Several other studies report no association between GSTT1 genotype and lung cancer risk (3744).
This study is the first to report increased risk of early-onset lung cancer in African Americans who carried the A/G or G/G genotypes at the GSTP1 locus. Of the GST genotypes explored in this analysis, GSTP1 polymorphisms are the least represented in the lung cancer literature. A pooled analysis of studies examining GSTP1 genotypes suggested increased risk of lung cancer associated with the G/G genotype (OR = 2.0, 95% CI 1.04.1, adjusted for age, hospital and tobacco smoking, A/A and A/G genotypes comprised the reference group) (41). However, the vast majority of subjects in these studies have been Caucasians without age of onset limitations and did not independently report statistically significant associations (4549). Investigating the relationships between smoking, GSTP1 genotype and lung cancer, Miller et al. (50) reported an interaction between GSTP1 G/G genotype and cumulative smoking exposure, with greater risks for current smokers than for exsmokers. In our study, risk associated with GSTP1 genotype in African Americans did not appear to be modified by smoking level. However, in our young-onset population, very few cases could be classified as exsmokers and the range of pack years reported among smokers was narrow.
GST combinations and their effects
There are limitations inherent in the single gene approach taken in most lung cancer association studies. The majority of cancers are regarded as complex diseases, which follow a polygenic model of inheritance. By examining risk associated with genotype combinations, our study acknowledges the complexity of the underlying mechanisms of lung cancer development and attempts to move data analysis in a direction better suited to identify the contributions of multiple genes.
Increased risk of lung cancer associated with GSTM1 null and GSTT1 null genotype combinations was identified in our population of Caucasian heavy smokers. Similar findings have been reported in Finnish squamous cell lung carcinoma cases (n = 44) who had >40 pack years of smoking (OR = 3.0, 95% CI 1.18.4) (45). Kelsey et al. (44) reported a 2.9-fold increase in risk in African Americans and Mexican Americans with this genotype combination after adjusting for smoking, age, sex and ethnicity (95% CI 1.17.7). Conversely, other studies did not observe this association (37,41,51,52). We did not find increased risk of early-onset lung cancer in those who jointly carry both GSTT1 and GSTP1 risk genotypes. This agrees with findings from several other populations, including Asian populations (38,52) and several European studies (39,41,45,47,48).
This paper reports the novel finding of an increased risk of lung cancer in African Americans who carry one or two risk GSTM1 and GSTP1 genes. Other epidemiological studies have also supported the relationship between increased risk of lung cancer and this genotype combination (5254). In particular, a study of younger age-at-onset individuals (55 years) found a 4-fold increase in risk of lung cancer in those who jointly carried the GSTP1 G/G genotype and GSTM1 null genotype after adjustment (OR = 4.03, 95% CI 1.4711.08) (25). However, other studies not limited to early-onset cases have not corroborated these findings (3840,45,48).
A doseresponse relationship was observed between the number of risk genotypes an individual carries and risk of early-onset lung cancer. The vast majority of research has focused on the joint effects of two genotype combinations, interactions with specific histology types or noted that stratified sample size limits sufficient statistical power (3942,45,55). While the functional role of GSTs in the carcinogen metabolism pathway has been established, the relative importance and combined functioning in conjunction with one another is yet to be determined. In this analysis of joint effects an assumption was made that each genotype confers a similar degree of risk.
Limitations found in most studies in lung cancer patients hold true in this study. The rapidly fatal nature of lung cancer makes obtaining biospecimens difficult and reduces the size of the population available for molecular epidemiology studies. During the first 4 years of subject accrual, DNA was not collected at the time of the interview. When a biospecimen was requested several years later, only 52 of 126 cases (50.1%) had archived tissue available and only 52 of 126 (41.3%) of controls were able to provide a sample. When DNA was requested at the time of the interview, 55.3% of controls agreed to give a sample and 51.1% of cases gave a blood or buccal sample or had archived tissue available. Those who did participate have genotype and allele frequencies comparable to published work (56,57). Additionally, there were no differences in smoking, age or family history of lung cancer between cases who did or did not provide a biospecimen (P = 0.84, 0.43 and 0.85, respectively). Among controls, those providing a biospecimen were slightly older (mean age = 41.1 versus 39.2, P < 0.0001) than those who did not, but there were no statistically significant differences in smoking (P = 0.11) or family history (P = 0.10).
Given the difficulty in collecting biospecimens, coupled with the relatively low frequency of certain polymorphisms, some ORs are based on small sample sizes. Thus, the results presented may not provide an accurate estimate of the true ORs. Sample size limits the ability to study geneenvironment interactions. Unknown or known but unmeasured confounders and effect modifiers (e.g. chemical, hormonal or dietary factors), may be partially responsible for some of the associations identified.
Detailed information for smoking, the overwhelming risk factor for lung cancer, was collected from all participants and the use of pack years, a continuous variable, results in less residual confounding than categorizing smoking into a two level, ever/never variable (58). Additionally, smokers were stratified into light and heavy categories by race, to account for the differences in smoking patterns seen between Caucasians and African Americans. Lower levels of cumulative amount smoked (i.e. pack years) reported by African Americans compared with Caucasians are consistent with previous local and national reports on tobacco use (59,60). The possible excess in risk in African American light smokers and Caucasian heavy smokers is likely to reflect the greater sample sizes and therefore power in these strata. However, the data may also suggest susceptibility to lung cancer differs by levels of exposure to tobacco smoke. The greater effect of metabolic polymorphisms at lower levels of tobacco exposure have been identified in lung cancer, particularly for cytochrome P-450 1A1 (CYP1A1) polymorphisms (61,62). The evidence suggesting a differential risk associated with GST genotypes, amount of tobacco exposure and lung cancer risk is not clear, with some studies supporting greater effects at lower levels of exposure (63,64), and others finding the opposite (65,66). In our population, significant interactions between pack years of smoking and GSTM1 and pack years of smoking and GSTT1 were identified in Caucasians. These findings may lend support to the theory that susceptibility to lung cancer is moderated by both tobacco exposure and genotype.
Our study had several important strengths. Cases and controls were both population-based and included a sufficient number of African Americans to estimate risk of lung cancer in this under-studied population. The focus on early-onset disease and the African American population, where genetic susceptibility might be greater, is unique in the current GST literature (33,67). Stratifying analyses by race decreased the possibility of population stratification biasing the results. Our ability to examine and adjust for known confounders, collected through structured interviews, is an additional strength of this study. In addition, incident cases were identified quickly after diagnosis, which limits survival bias. This may prove to be especially important if these GSTs play a role in response to treatment and subsequent survival.
This study focused on cases diagnosed before 50 years of age, well below the median age of onset for lung cancer (70 years for Caucasians and 66 years for African Americans) (68). Although early-onset is arbitrarily defined, younger age of onset is suggestive of increased genetic susceptibility to lung cancer. Familial aggregation of disease has been more strongly identified in younger age groups, suggesting a heritable component to lung cancer (6972). More recently, linkage to a region on chromosome 6 has been described in familial lung cancer adding evidence for the role of genetic susceptibility for this disease, particularly when age of onset is young (73).
Our results suggest that specific combinations of GSTM1, GSTT1 and GSTP1 are involved in lung cancer susceptibility in early-onset cases. However, larger sample sizes are needed to confirm these findings and clarify differences associated with other modifiers of early age of onset lung cancer risk. Joint analysis of genes involved in carcinogen metabolism pathways may identify individuals who are at higher risk of developing lung cancer with a greater certainty than single gene studies.
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Acknowledgments |
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References |
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