GSTM1 null and NAT2 slow acetylation genotypes, smoking intensity and bladder cancer risk: results from the New England bladder cancer study and NAT2 meta-analysis

Abstract

Associations between bladder cancer risk and NAT2 and GSTM1 polymorphisms have emerged as some of the most consistent findings in the genetic epidemiology of common metabolic polymorphisms and cancer, but their interaction with tobacco use, intensity and duration remain unclear. In a New England population-based case–control study of urothelial carcinoma, we collected mouthwash samples from 1088 of 1171 cases (92.9%) and 1282 of 1418 controls (91.2%) for genotype analysis of GSTM1, GSTT1 and NAT2 polymorphisms. Odds ratios and 95% confidence intervals of bladder cancer among New England Bladder Cancer Study subjects with one or two inactive GSTM1 alleles (i.e. the ‘null’ genotype) were 1.26 (0.85–1.88) and 1.54 (1.05–2.25), respectively (P-trend = 0.008), compared with those with two active copies. GSTT1 inactive alleles were not associated with risk. NAT2 slow acetylation status was not associated with risk among never (1.04; 0.71–1.51), former (0.95; 0.75–1.20) or current smokers (1.33; 0.91–1.95); however, a relationship emerged when smoking intensity was evaluated. Among slow acetylators who ever smoked at least 40 cigarettes/day, risk was elevated among ever (1.82; 1.14–2.91, P-interaction = 0.07) and current heavy smokers (3.16; 1.22–8.19, P-interaction = 0.03) compared with rapid acetylators in each category; but was not observed at lower intensities. In contrast, the effect of GSTM1-null genotype was not greater among smokers, regardless of intensity. Meta-analysis of the NAT2 associations with bladder cancer showed a highly significant relationship. Findings from this large USA population-based study provided evidence that the NAT2 slow acetylation genotype interacts with tobacco smoking as a function of exposure intensity.

Introduction

The association between bladder cancer risk and polymorphisms in two carcinogen detoxification genes, NAT2 and GSTM1, has emerged as one of the most consistent associations in the genetic epidemiology of common polymorphisms and cancer. Tobacco smoking is an important risk factor for bladder cancer and previous analyses suggest that the relative risk from smoking is stronger for NAT2 slow acetylators than for rapid acetylators. Although this relationship has not always been demonstrated consistently, interaction is biologically plausible since aromatic amines, which are thought to be the most important class of bladder carcinogens in tobacco smoke, are detoxified by NAT2. Evidence to support this association includes a recent comprehensive meta-analysis that reported increased overall risk of bladder cancer with the GSTM1-null genotype, but only the NAT2 slow acetylator genotype showed evidence of an interaction with smoking, particularly with increasing smoking intensity (measured as cigarettes/day) (1–3). Currently, it is unclear whether the inconsistent associations observed across case–control studies are due to inadequate power to detect significant main effects or measurement error resulting from inadequate assessment of smoking status, including analysis of smoking intensity and/or consideration of the aromatic amine content and type (black versus blond) of tobacco consumed across populations.

We conducted a large population-based case–control study in three New England states (Maine, Vermont and New Hampshire) and collected mouthwash samples for DNA extraction among 1088 of 1171 urothelial carcinoma cases and 1282 of 1418 controls enrolled in this study. A total of 2370 DNA samples passed quality control and were successfully genotyped. This study was designed to have sufficient statistical power to investigate associations between the GSTM1, GSTT1 and NAT2 genotypes and bladder cancer risk, using a detailed questionnaire to collect comprehensive information on smoking habits that included duration, intensity and cessation of smoking (4). We also updated a meta-analysis of NAT2 variants, smoking and bladder cancer risk, including results for 7961 cases and 13 819 controls.

Materials and methods

Study population

The New England Bladder Cancer Study is a population-based study that was conducted in Maine, Vermont and New Hampshire. Cases, between 30 and 79 years of age, were newly diagnosed and histologically confirmed with urinary carcinoma of the bladder (including carcinoma in situ) between 1 September 2001 and 31 October 2004 (Maine and Vermont) or between 1 January 2002 and 31 July 2004 (New Hampshire) among residents of the three states. Rapid case ascertainment during the study period was conducted through hospital pathology departments and hospital and state cancer registries. We interviewed 1213 bladder cancer cases (65% of 1878 eligible cases). Control subjects aged 30–64 years were selected randomly from the Department of Motor Vehicle records in each state, and controls aged 65–79 years were selected from beneficiary records of the Centers for Medicare and Medicaid Services. Controls were frequency matched to cases by state, sex and age at case diagnosis within 5 years. Additional details of the New England Bladder Cancer Study population have been previously reported (4). We interviewed 1418 (594 Department of Motor Vehicle and 824 Centers for Medicare and Medicaid Services) controls (65% of eligible Department of Motor Vehicle and 65% of eligible Centers for Medicare and Medicaid Services controls). Participants were interviewed at home by trained interviewers using a detailed computer-assisted personal interview to obtain information on demographics, occupational and residential histories, fluid intake, use of tobacco and hair coloring products, family history of cancer, medication use and dietary factors.

We defined ‘never-smokers’ as subjects who had smoked <100 cigarettes over their lifetime. ‘Occasional smokers’ reported that they had smoked >100 cigarettes overall, but never consumed cigarettes regularly (i.e. at least one cigarette/day for at least 6 months). ‘Regular smokers’ were categorized as ‘former smokers’ if they had quit smoking ≥1 year before the reference date (i.e. diagnosis date for cases, selection date for controls) or ‘current smokers’ if they were still smoking regularly at the time of their interview or had quit within 1 year of the reference date.

Genotyping analyses were successfully conducted on 1088 of 1171 (92.9%) cases and 1282 of 1418 (91.2%) controls who provided a mouthwash sample for genomic DNA extraction and genetic analyses. A total of 2458 of 2589 samples tested (94.9%) passed quality control and of these 2370 were successfully genotyped (96.4%). Genotyped subjects did not differ by age, state, sex or smoking characteristics, from those included in the case–control study as a whole.

Procedures

DNA for genotyping was extracted from exfoliated buccal cells collected from mouthwash samples, using standard phenol–chloroform extraction methods. Genotypes were analyzed at the Core Genotyping Facility of the Division of Cancer Epidemiology and Genetics, of the National Cancer Institute. For the GSTM1 and GSTT1 assays, melt curve/copy number assays (Applied Biosystems, Foster City, CA) were used to determine the number of deleted copies (0, 1, 2) of each gene. For NAT2 single-nucleotide polymorphisms (SNPs), a TaqMan assay (Applied Biosystems) was used. Descriptions and methods for each assay can be found at the National Cancer Institute SNP500Cancer website (SNP500cancer.nci.nih.gov). Genotype assays used to determine acetylation status for NAT2 included the following SNPs: (rs1208, K268R A > G; rs179993, G286E G > A; rs1041983, Y94Y C > T; rs1801280, I114T T > C; rs1799929, L161L C > T; rs1799930, R197Q G > A and rs1805158, R64Q G > A). All genotypes were in Hardy–Weinberg equilibrium among the control population. Duplicate quality control samples showed 100% agreement for all assays. Completion rates for NAT2 SNPs were ≥99% except for rs1799931 which was 98%, and GSTM1 and GSTT1 which were 98 and 93%, respectively.

The NAT2 SNP results were used to assign the most probable functional acetylation genotypes previously identified and defined in human populations (5,6). Individuals homozygous for NAT2 rapid acetylator alleles (NAT2*4 (ref), NAT2*12A [rs1208; Ex2-367 (803A > G)], NAT2*13A [rs1041983; Ex2 +288C > T (282C > T)]) were classified as having the rapid acetylator genotype. Individuals homozygous for slow acetylator alleles (NAT2*5A [rs1801280; Ex2 +347 T > C (341T > C) and rs1799929; Ex2 +487C > T (481C > T)], NAT2*5B [rs1801280; Ex2 +347 T > C (341T > C) & rs1799929; Ex2 +487C > T (481C > T) and rs1208; Ex2-367 (803A > G)], NAT2*5C [rs1801280; Ex2 +347 T > C (341T > C) and rs1208; Ex2-367 (803A > G)] NAT2*6A [rs1041983; Ex2 +288C > T (282C > T) and rs1799930; Ex2 -580G > A (590G> A)], NAT2*7B [rs1041983; Ex2 +288C > T (282C > T) and rs1799931; Ex2 -313G > A (857 G > A)]) were classified as having a slow acetylator genotype. Heterozygous individuals (one rapid and one slow NAT2 allele) were classified as having an intermediate acetylator genotype. GSTM1 and GSTT1 genotypes were defined as null (−/−) if a deletion was present in both copies of the gene and active if one (+/−) or two (+/+) copies of the gene were present.

Statistical analysis

We computed odds ratios (ORs) and 95% confidence intervals (CIs) from unconditional logistic regression models, adjusting for age (<55, 55–64, 65–74, 75+ years), sex, state (Maine, New Hampshire and Vermont), smoking status (never and regular), quitting status of regular smokers (former and current), smoking duration (<40 and ≥40 years) and smoking intensity (0.5–20, 21–39 and ≥40 cigarettes/day). Occasional smokers were dropped from analyses of regular smokers. Interactions between genotypes and tobacco exposure variables were investigated using a likelihood ratio test to compare models with and without interaction terms. The assumption of gene–environment independence was satisfied among the controls. Likelihood ratio tests for multiplicative interaction were used to assess whether the genotype ORs within categories stratified by smoking habits differed significantly from each other or whether smoking ORs within genotype categories differed significantly from each other. The global P-value from the likelihood ratio test was calculated to evaluate overall heterogeneity between logistic models with and without interaction terms.

We conducted a HuGE Navigator literature database search and a PubMed literature search of peer reviewed studies of NAT2 acetylation status and bladder cancer, published on or before July 2009 that were written in English. A random-effect model was used to estimate summary ORs and 95% CIs by weighting each study result by a within- and between-study variance (7). Homogeneity of study results was assessed by the I-squared statistic (I²) (8). Harbord’s test of the relationship between the magnitude of the association and the precision of the risk estimate was used to identify publication or related biases (P = 0.20 for NAT2) (9).

A case-only design was used in the meta-analysis to assess the presence of a multiplicative interaction between NAT2 genotype and smoking status (regular/never) and interaction odds ratios (OR_int) and 95% CIs were calculated. This approach was used because it allowed inclusion of studies lacking information on the cross-classification of genotype and smoking status among controls. In addition, it removed possible biases resulting from the inclusion of hospitalized controls with diseases related to tobacco use, and it is a powerful design to test for multiplicative interactions under the assumption of independence of the NAT2 genotype with smoking status in the population (10,11) . We also performed a case-only meta-analysis using data from three other USA studies including the current one that provided pack-year intensity information (12–14). We were able to define heavy smokers across studies when they were consistently defined among regular smokers with at least 28 pack-years of exposure. Statistical analyses were done with STATA (Version 9.1, Special Edition; STATA Corporation, College Station, TX).

Results

The study population was predominantly Caucasian, male, and included past or current smokers (Table I). The controls were very well matched to the cases on age, sex, state and race (Table I). Increased risk of urothelial cancer was observed among regular smokers but not among occasional smokers (i.e. those who had smoked >100 cigarettes overall, but never consumed at least one cigarette/day for at least 6 months) compared with never-smokers. Increased bladder cancer risks were observed with all categories of smoking by duration and cigarettes/day. Risks estimated by cigarettes/day appeared to plateau in the highest categories.

Table I.

Characteristics of study population in the New England Bladder Cancer Study

Demography	Cases	(%)	Controls	(%)	OR-crude (95% CI)	P-value
Sex
Female	252	23.2	335	26.1
Male	836	76.8	947	73.9
Race
White	1023	94.1	1212	94.7
Native American/White	55	5.1	51	4.0
Others	9	0.8	17	1.3
Age
30–54	176	16.2	233	18.2
55–64	285	26.2	300	23.4
65–74	403	37.0	500	39.0
75–79	224	20.6	249	19.4
Education
<High school	233	21.4	209	16.3
High school graduate	337	31.0	373	29.1
Vocational	87	8.0	103	8.0
Some college	162	14.9	209	16.3
College graduate	144	13.2	192	15.0
Postgraduate	125	11.5	196	15.3
State
Maine	528	48.5	673	52.5
Vermont	184	16.9	209	16.3
New Hampshire	376	34.6	400	31.2
Smoking status
Never	160	14.7	426	33.3	1.00 (REF)
Occasional	22	2.0	34	2.7	1.73 (0.98–3.06)	0.06
Former	560	51.5	636	49.7	2.34 (1.89–2.90)	<0.0001
Current	345	31.7	185	14.4	4.97 (3.85–6.41)	<0.0001
Duration of smoking
<10–19	162	14.9	271	21.1	1.59 (1.22–2.07)	0.001
20–29	174	16.0	200	15.6	2.32 (1.76–3.04)	<0.0001
30–39	218	20.0	141	11.0	4.21 (3.11–5.44)	<0.0001
≥40	347	31.9	201	15.7	4.60 (3.57–5.91)	<0.0001
Cigarettes/day
0.5–19	210	19.7	248	19.9	2.25 (1.74–2.92)	0.001
20–29	395	37.1	324	26.0	3.25 (2.57–4.10)	<0.0001
30–39	126	11.8	92	7.4	3.65 (2.64–5.04)	<0.0001
≥40	174	16.3	156	12.5	2.97 (2.24–3.94)	<0.0001

Having at least one GSTM1-null allele was associated with an increased risk of urothelial carcinoma of the bladder; the ORs and 95% CIs for individuals lacking one and two copies of the GSTM1 gene were 1.26 (0.85–1.88) and 1.54 (1.05–2.25, P-trend = 0.008), respectively (Table II). In contrast, absence of at least one GSTT1 allele was unrelated to risk (OR = 1.15; 0.89–1.48). None of the six individual NAT2 SNPs were associated with increased risk. Similarly, subjects with the NAT2 slow acetylation status that was estimated from combinations of these SNPs was not associated with bladder cancer risk compared with subjects with rapid/intermediate acetylation status (OR = 1.04; 0.82–1.28).

Table II.

Bladder cancer risk associated with genotypes, OR and 95% CI

Genotype	Case	%	Control	%	Adjusteda OR (95% CI)	P-value
GSTM1
Active/active	70	6.65	107	8.66	1.00 (REF)
Active/null	330	31.34	438	35.47	1.26 (0.85–1.88)	0.24
Null/null	653	62.01	690	55.87	1.54 (1.05–2.25)	0.02
Any active versus null	400	37.99	545	44.13	1.28 (1.04–1.57)	0.02
Per allele, P-trend					1.23 (1.23–1.44)	0.008
GSTT1
Active/active	339	33.76	394	33.42	1.00 (REF)
Active/null	455	45.32	548	46.48	0.92 (0.73–1.16)	0.49
Null/null	210	20.92	237	20.1	1.10 (0.82–1.46)	0.52
Any active versus null	794	79.08	942	79.90	1.15 (0.89–1.48)	0.28
Per allele, P-trend					1.03 (0.90–1.19)	0.66
NAT2
rs1208; Ex2-367 A>G; K268R
AA	338	31.21	401	31.65	1.00 REF
AG	532	49.12	602	47.51	1.01 (0.81–1.27)	0.90
GG	213	19.67	264	20.84	0.88 (0.66–1.17)	0.38
rs1799931; Ex2 -313G>A; G286E
GG	1020	93.92	1209	95.2	1.00 (REF)
AG	66	6.08	61	4.8	1.22 (0.79–1.87)	0.37
rs1041983; Ex2 +288C>T; Y94Y
CC	494	45.53	604	47.52	1.00 (REF)
CT	482	44.42	537	42.25	1.12 (0.91–1.38)	0.28
TT	109	10.05	130	10.23	1.17 (0.83–1.65)	0.35
rs1801280; Ex2 +347T>C; I114T
TT	314	28.97	383	30.11	1.00 (REF)
TC	538	49.63	606	47.64	1.04 (0.83–1.31)	0.71
CC	232	21.4	283	22.25	0.89 (0.67–1.18)	0.41
rs1799929; Ex2 +487 C>T; L161L
CC	323	29.77	416	32.68	1.00 REF
CT	549	50.6	590	46.35	1.15 (0.91–1.44)	0.24
TT	213	19.63	267	20.97	0.91 (0.69–1.21)	0.52
rs1799930; Ex2 -580 G>A; R197Q
GG	539	49.72	641	50.47	1.00 (REF)
GA	455	41.97	523	41.18	1.03 (0.84–1.26)	0.79
AA	90	8.3	106	8.35	1.23 (0.85–1.79)	0.27
Acetylation status
Rapid	42	3.89	59	4.67	1.00 (REF)
Intermediate	382	35.34	451	35.68	0.92 (0.54–1.58)	0.77
Slow	657	60.78	754	59.65	0.97 (0.57–1.65)	0.91
Rapid + intermediate versus Slow	1039	96.12	1205	95.33	1.04 (0.82–1.28)	0.67
Per allele, P-trend					1.03 (0.86–1.33)	0.76

^aAdjusted for sex, age categories (4), state (ME,VT and NH), smoking status (never and regular) and duration of smoking (<10,10–19,20–39 and 40+) in years.

The association between GSTM1 genotype and bladder cancer was similar by smoking status (Table III). The GSTT1-null genotype, when compared with the combined active genotypes, was inversely related to bladder cancer risk among never-smokers but the tests for trend (P = 0.28) and interaction with smoking status were not significant. ORs were not elevated among NAT2 slow acetylators compared with rapid acetylators (OR = 1.42; 0.53–3.79, P = 0.49) and rapid/intermediate acetylators (OR = 1.33; 0.91–1.95, P = 0.14) among current smokers, and multiplicative interactions were not statistically significant (P-interaction = 0.09 and 0.12, respectively).

Table III.

Bladder cancer risk associated with genotypes, stratified by smoking status

Genotype	Cases/controls	Never smokersa		Former smokersab			Current smokersab			P-interaction
		OR (95% CI)	P-value	Cases/controls	Adjusted OR (95% CI)	P-value	Cases/controls	Adjusted OR(95% CI)	P-value
GSTM1
Active/Active	10/32	1.00 (REF)		38/61	1.00 (REF)		21/13	1.00 (REF)
Active/Null	42/155	0.84 (0.38–1.86)	0.67	177/204	1.42 (0.89–2.26)	0.14	104/68	0.93 (0.43–1.99)	0.86
Null/Null	101/229	1.37 (0.65–2.92)	0.41	399/345	1.61 (1.04–2.51)	0.03	210/96	1.40 (0.66–2.97)	0.38
Any active versus null		1.58 (1.07–2.34)	0.02		1.22 (0.96–1.56)	1.10		1.48 (1.01–2.17)	0.05	0.48
Per allele, P-trend		1.37 (1.00–1.88)	0.05		1.22 (1.01–1.46)	0.04		1.31 (0.97–1.77)	0.07	0.75
GSTT1
Active/active	49/130	1.00 (REF)		172/199	1.00 (REF)		112/52	1.00 (REF)
Active/null	84/179	1.22 (0.80–1.87)	0.36	228/270	0.99 (0.75–1.30)	0.94	132/84	0.83 (0.53–1.30)	0.42
Null/null	22/87	0.65 (0.36–1.16)	0.14	109/114	1.10 (0.78–1.54)	0.60	73/32	1.12 (0.65–1.93)	0.69
Any active versus null		0.57 (0.34–0.96)	0.04		1.10 (0.82–1.49)	0.52		1.24 (0.77–2.01)	0.37	0.70
Per allele, P-trend		0.86 (0.66–1.13)	0.28		1.04 (0.88–1.23)	0.65		1.03 (0.79–1.35)	0.82	0.40
Acetylation status
Rapid	8/24	1.00 REF		23/21	1.00 (REF)		10/10	1.00 (REF)
Intermediate	55/148	1.19 (0.50–2.83)	0.68	203/223	0.82 (0.43–1.55)	0.54	112/70	1.07 (0.40–2.90)	0.89
Slow	96/251	1.21 (0.52–2.80)	0.66	331/378	0.79 (0.42–1.48)	0.46	220/104	1.42 (0.53–3.79)	0.49
Rapid + intermediate versus slow		1.04 (0.71–1.51)	0.84		0.95 (0.75–1.20)	0.66		1.33 (0.91–1.95)	0.14	0.12
Per allele, P-trend		1.05 (0.77–1.43)	0.74		0.94 (0.76–1.15)	0.53		1.27 (0.92–1.77)	0.15	0.09

^aAdjusted for sex, age categories(4), state (ME, NH,VT).

^bAdjusted for duration of smoking (<40, ≥40 years).

In Table IV, risks associated with smoking and genotype are presented in two ways, first by the risk estimate for the NAT2 slow compared with the rapid/intermediate acetylation genotype within each smoking stratum and as joint associations of NAT2 and smoking using never smokers with the rapid/intermediate acetylation genotype as a common referent. There was no evidence of increased risk among former or current smokers among slow compared with rapid/intermediate acetylators. However, when we incorporated smoking intensity, bladder cancer risk was significantly elevated among slow acetylators who were the heaviest smokers (i.e. those reporting having smoked at least 40 cigarettes/day) (OR = 1.82; 1.14–2.91, P = 0.01, P-interaction = 0.07) but a general pattern of enhancement of smoking effects for NAT2 acetylation status across the lower intensity categories was not observed. When analyses were restricted to current smokers, the OR was elevated further among NAT2 slow acetylators who smoked at least 40 cigarettes/day compared with current smokers with the rapid/intermediate genotype and the interaction was statistically significant (OR = 3.16; 1.22–8.19, P = 0.02, P-interaction = 0.03) but again, a pattern of enhancement of smoking effects for NAT2 acetylation status across the lower intensity categories among current smokers was not observed. In the joint analysis, the OR among current smokers of at least 40 cigarettes/day with the slow acetylation genotype was significantly elevated (OR = 7.12; 3.23–15.7) when compared with never-smokers with the rapid/intermediate genotype as a common referent. Modification by NAT2 acetylation status and smoking duration among regular smokers was not observed.

Table IV.

Smoking chracteristics, the NAT2 acetylation genotype and bladder risk

Smoking characteristics	Frequency				OR (95% CI) for NAT2 slow genotype association by smoking characteristica	OR (95% CI) for joint NAT2 genotypes			P-value interactiona
	NAT2 rapid/intermediate		NAT2 slow			NAT2 rapid/intermediate		NAT2 slow
	Case	Controls	Case	Controls	OR (95% CI)	P-value	OR (95% CI)	OR (95% CI)
Smoking statusb
Never	63	172	96	251	1.04 (0.71–1.51)	0.84	1.00 (REF)	1.05 (0.72–1.53)
Ever	360	338	561	502	1.04 (0.86–1.26)	0.68	2.90 (2.10–4.02)	3.04 (2.22–4.16)	0.99
Regular
Former	226	244	331	378	0.95 (0.75–1.20)	0.66	2.44 (1.74–3.45)	2.32 (1.67–3.22)	0.63
Current	122	80	220	104	1.35 (0.93–1.96)	0.11	4.32 (2.88–6.49)	5.85 (4.03–8.50)	0.35
Cigarettes/dayc
Never							1.00 (REF)	1.04 (0.72–1.52)
0.5–<20	84	98	125	141	1.01 (0.68–1.49)	0.97	1.82 (1.20–2.78)	1.88 (1.27–2.76)	0.90
20–29	159	129	233	190	1.00 (0.73–1.36)	0.98	2.45 (1.67–3.60)	2.43 (1.70–3.49)	0.85
30–39	52	30	74	62	0.60 (0.34–1.07)	0.09	3.72 (2.15–6.42)	2.17 (1.36–3.46)	0.12
≥40	54	67	117	88	1.82 (1.14–2.91)	0.01	1.52 (0.94–2.45)	2.85 (1.89–4.31)	0.07
P-global									0.06
Smoking duration (years)d							1.00 (REF)	1.05 (0.72–1.53)
<40	223	234	328	363	0.95 (0.74–1.20)	0.66	1.94 (1.32–2.83)	1.82 (1.26–2.63)	0.68
≥40	123	88	220	113	1.40 (0.98–2.00)	0.07	2.16 (1.35–3.46)	3.00 (1.93–4.67)	0.26
P-global									0.20
Current smokers
Cigarettes/dayc
Never							1.00 (REF)	1.04 (0.71–1.51)
0.5–<20	26	23	39	29	1.05 (0.49–2.28)	0.75	2.53 (1.27–5.04)	2.80 (1.48–5.29)	0.97
20–29	56	36	108	43	1.60 (0.91–2.78)	0.10	3.27 (1.81–5.92)	5.20 (3.04–8.90)	0.21
30–39	21	5	33	20	0.34 (0.11–1.05)	0.06	9.67 (3.37–27.7)	3.04 (1.52–6.10)	0.07
≥40	20	16	39	11	3.16 (1.22–8.19)	0.02	2.23 (1.00–5.00)	7.12 (3.23–15.7)	0.03
P-global									0.03
Smoking duration (years)d
Never							1.00 (REF)	1.05 (0.72–1.53)
<40	46	35	76	47	1.18 (0.67–2.11)	0.56	2.10 (1.41–3.13)	1.90 (1.30–2.79)	0.71
≥40	76	44	144	57	1.44 (0.89–2.34)	0.14	2.37 (1.37–4.11)	3.08 (1.86–5.11)	0.30
P-global									0.58

^aFor differences between the OR for NAT2 slow compared with rapid-acetylation genotype within strata defined by smoking characteristics.

^bORs are from conventional logistic regression models adjusted for sex, age and state.

^cCigarettes/day: ORs are from conventional regression models adjusted for age, sex, region, smoking cessation (current/former) and smoking duration (<40 and ≥40 years).

^dSmoking duration: ORs are from conventional regression models adjusted for age, sex, region, smoking cessation (current/former) and cigarettes/day (0.5–20, 21–39 and ≥40).

To place this analysis in the context of other published studies, we updated a previous meta-analysis of NAT2 acetylation status (1,3), with an additional 10 studies (14–22) including the current study (N = 46), for a total of 7961 cases and 13 819 controls (Figure 1). Overall, the summary risk estimate was significantly elevated (OR = 1.37; 1.24–1.52, P = 1.17 × 10⁻⁹) for slow compared with rapid/intermediate acetylators and there was statistical evidence of heterogeneity between studies (P = 0.001), but no evidence of publication or related bias (Harbord’s test P = 0.22). When stratified by geographic region, heterogeneity remained between European and USA Caucasian studies but was no longer observed between European Caucasian and Asian studies or between Asian and USA Caucasian studies (P = 0.003, data not shown). Within-region heterogeneity was observed only in Asian studies (I² P-value = 0.02) and remained when analyses were limited to studies of at least 100 cases (I² P-value = 0.02). The relative risk associated with NAT2 slow acetylation status was strongest for European Caucasian (OR = 1.43; 1.30–1.57, P = 4.8 × 10⁻¹⁴) and Asian studies (OR = 1.53; 1.11–2.10, P = 0.01). With the addition of data from the current New England study and recent studies based in Los Angeles, CA (14) and Houston, TX (18), the association between bladder cancer risk and NAT2 slow acetylation in USA Caucasians was weaker than that observed in European Caucasians (OR = 1.11; 0.96–1.29, P = 0.15) and remained so when analyses were restricted to studies of 100 cases or more (OR = 1.12; 0.97–1.29, P = 0.13). An updated case-only meta-analysis of NAT2 acetylation status, smoking, and bladder cancer of 27 studies and 6513 cases is presented in Figure 2. For all ethnicities, we observed a significant (multiplicative) OR_int of 1.22 (1.07–1.40, P = 0.003), without evidence of heterogeneity (I² P-value = 0.51). After stratification by geographic region, the OR_int was significantly elevated only in European Caucasians (OR_int = 1.38; 1.13–1.68, P = 0.002). With the addition of our New England data and that from two recent USA studies (14,18), the summary OR_int estimate in USA populations did not reach statistical significance, although it was higher than that estimated in a previous report (OR_int = 1.15; 0.92–1.45, P = 0.23) versus (OR_int = 0.97; 0.67–1.39, P = 0.85) (3). The main effect of NAT2 slow acetylation status and bladder cancer risk was significantly elevated in Asian studies (Figure 1), but an interaction with smoking was not observed (OR_int = 0.99; 0.62–1.60, P = 0.97) and was similar when analyses were limited to studies of at least 100 cases.

Fig. 1.

Meta-analysis of NAT2 slow acetylation genotype and bladder cancer risk, overall and stratified by geographic region.

Fig. 2.

Case-only meta-analysis of the interaction between NAT2 slow acetylation genotype, smoking and bladder cancer risk, overall and stratified by geographic region.

To investigate the hypothesis that an NAT2 acetylation/smoking interaction may be relevant only among the heaviest smokers, a case-only meta-analysis was conducted among USA studies that reported risk among smokers by pack-years of exposure (12–14) (Figure 3). A significant interaction with NAT2 genotype was revealed among smokers with at least 28 pack-year of exposure (OR_int = 1.33; 1.01–1.77, P = 0.05). Heterogeneity across studies was not observed (I² P-value = 0.21).

Fig. 3.

Case-only meta-analysis of the interaction between NAT2 acetylation genotype, smoking and bladder cancer in USA studies of subjects who reported having at least 28 pack-years of exposure.

Discussion

The results of this large case–control study conducted in New England provide additional evidence of an association between GSTM1 genotype and risk of bladder cancer. We applied new copy number assays for GSTM1 and GSTT1 that allowed discrimination between individuals with one or two active copies of each gene. Using subjects with two active copies as a common referent group provided more precise risk estimates for subjects lacking one or two active alleles and the ability to calculate risk per allele. We observed a significant association between the GSTM1-null genotype and bladder cancer risk overall and a significant positive trend with the number of null alleles present. Overall, the GSTM1 homozygous null genotype was associated with a 28% increased risk when compared with individuals with at least one active copy and 54% when compared with individuals with two active copies of the gene. The risk estimates observed in this study are similar to a previous Spanish study that employed a similar assay (1,3). In this study, we did not observe significant associations between GSTT1 or NAT2 genotypes and bladder cancer risk as main effects, although the estimate among NAT2 slow acetylators was non-significantly elevated among current smokers compared with rapid/intermediate acetylators. With evaluation of smoking intensity, a statistically significant association emerged between NAT2 slow acetylation genotype and bladder cancer risk, but only among regular smokers (ever and current) who had smoked at least 40 cigarettes/day in a model adjusted for smoking duration, quitting status and other covariates. Although significantly elevated among the heaviest smokers, there was no evidence of a general pattern of increasing enhancement of smoking effects for the NAT2 slow acetylation genotype among individuals in the lower intensity categories. In USA studies that reported pack-years of exposure, we observed a positive association among the heaviest smokers. Additional studies are needed to explain the heterogeneity observed between geographic regions, which could be explained in part by differences in the aromatic amine content of different types of tobacco (23,24).

Other studies including our own support the hypothesis that the NAT2 genotype modifies bladder cancer risk as a function of smoking intensity, rather than duration (1,2,13,14). In a bladder cancer study conducted in Spain, Lubin et al. (2) observed that variation in smoking risk by NAT2 acetylation status resulted from an interaction with smoking intensity, rather than pack-years. The heterogeneity associated with genotype suggests that among slow acetylators, detoxification of the aromatic amines present in cigarette smoke through acetylation may become saturated at higher smoking intensity, thus minimizing the beneficial NAT2 detoxification that is available to slow acetylator genotypes compared with rapid/intermediate acetylator genotypes. As shown in our meta-analysis (Figure 3), studies that evaluated and reported pack-years of exposure [e.g. Gu et al. (13)] have observed that the NAT2 slow acetylation genotype was associated with bladder cancer risk overall (OR = 1.31; 1.01–1.70); risk was greater among heavy smokers (at least 28 pack-years of exposure) (OR = 2.11; 1.33–3.55) than among light smokers (OR = 0.96; 0.61–1.53) and the interaction between NAT2, pack-years of smoking and bladder cancer risk was statistically significant. In Yuan et al. (14), an association between NAT2 genotype and bladder cancer was not observed overall; however, when pack-years were considered, bladder cancer risk was significantly elevated among slow acetylators with at least 30 pack-years (OR = 1.51; 0.99–2.29), but not among those with <30 pack-years of exposure (OR = 0.92; 0.61–1.39) compared with rapid/intermediate acetylators with the same rate of exposure. Similarly, in this New England population, bladder cancer risk was significantly elevated only among slow acetylators who smoked at least 40 cigarettes/day. The interaction was significant among regular (ever and current) smokers and was strengthened in analyses restricted to current smokers. In contrast, Taylor et al. (12) observed neither a main effect of NAT2 nor an interaction among heavy smokers (smokers with >35 pack-years of exposure). However, this study was small and the association could be due to chance. Additional studies of genetic variation with detailed assessment of both tobacco type and smoking intensity will be required to further elucidate this relationship.

Lastly, unlike NAT2 slow acetylation, the risk associated with the GSTM1-null genotype in this study was not higher among smokers and appears to protect against bladder cancer regardless of smoking status. An explanation for this finding remains to be elucidated since the GSTM1 enzyme is known to conjugate several carcinogenic compounds in tobacco smoke such as polycyclic aromatic hydrocarbons that can also be found as occupational and environmental exposures (25). GSTM1 can also protect from oxidative damage through metabolism of reactive oxygen species (26) and has also been shown to mediate detoxification through methylation of carcinogens such as arsenic (27). Overall, however, these findings suggest that our understanding of its mode of action for detoxification of carcinogens may be incomplete.

The New England Bladder Cancer Study is a large well-designed population-based study with detailed data on smoking habits, prior tobacco consumption patterns and high-quality genetic data on NAT2 SNPs known to account for functional variation in acetylation status in Caucasians. We also applied new copy number assays for GSTM1 and GSTT1 analysis that were able to distinguish subjects with one or two active alleles. This assay had higher completion and concordance rates over those used in the past (1). A limitation of the case-control study is the 65% participation rate among both case patients and controls. The non-differential nonresponse rate of 35% may have lead to an underestimation of some risks estimated but would be unlikely to confound our findings because by definition, it would require association with both genotype and case–control status, which it is not. With the addition of 2370 USA cases and controls, this study was sufficiently large to detect weak associations between genetic variants and bladder cancer risk; however, it was still limited in terms of detecting gene–exposure interactions.

In summary, results from this case–control study provide additional evidence that GSTM1 genotype is associated with bladder cancer risk and that the NAT2 genotype interacts with smoking intensity. Additional large investigations of bladder cancer risk, NAT2 genetic variation and comprehensive assessment of smoking habits, tobacco types and aromatic amine content of tobacco will be needed to further evaluate these relationships.

Funding

Intramural Research Program of the National Institutes of Health, National Cancer Institute, Division of Cancer Epidemiology and Genetics, Occupational and Environmental Epidemiology Branch.

Glossary

Abbreviations

CI

confidence interval

OR

odds ratio

OR_int

interaction odds ratio

SNP

single-nucleotide polymorphism