CYP2C9 Variants Increase Risk of Colorectal Adenoma Recurrence and Modify Associations with Smoking but Not Aspirin Treatment

Abstract

Purpose

The cytochrome P450 2C9 enzyme (CYP2C9) is involved in metabolism of endogenous compounds, drugs and procarcinogens. Two common nonsynonymous polymorphisms in CYP2C9 are associated with reduced enzyme activity: CYP2C9*2 (rs1799853, R144C) and CYP2C9*3 (rs1057910, I359L).

Methods

We investigated whether CYP2C9 genotype was associated with risk of colorectal adenoma and/or modified associations with aspirin treatment or cigarette smoking in a cohort of 928 participants in a randomized trial of aspirin chemoprevention. Generalized linear regression was used to compute relative risks (RRs) and 95% confidence intervals (95% CIs). Multiplicative interactions terms were used to assess effect modification.

Results

CYP2C9 genotype was associated with increased risks for adenoma recurrence of 29% (RR=1.29, 95% CI =1.09–1.51) for ≥1 variant allele (CYP2C9*2 or *3) and 47% (RR=1.47, 95% CI=1.19–1.83) for ≥1 CYP2C9*3 allele. The risk for advanced lesions or multiple (≥3) adenomas was increased by 64% (RR=1.64, 95% CI=1.18–2.28) for ≥1 variant allele (CYP2C9*2 or *3) and 79% (RR=1.79, 95% CI=1.16–2.75) for ≥1 CYP2C9*3 allele. Genotype modified associations with smoking, but not aspirin treatment. The adenoma risk was increased by 26% (RR=1.26, 95% CI=0.99–1.58) for former smokers and 60% (RR=1.60, 95% CI=1.19–2.15) for current smokers among wild-type individuals, but there was no increased risk among individuals with ≥1 variant allele (CYP2C9*2 or *3) (P_interaction=0.04).

Conclusions

Carriers of CYP2C9 variants with lower enzyme activity have increased overall risk of colorectal adenoma but reduced adenoma risk associated with cigarette smoking. These results may be due to effects on the synthesis of endogenous eicosanoids and/or reduced activation of procarcinogens in smoke by CYP2C9 variants.

Introduction

As the second leading cause of cancer deaths in the United States, colorectal cancer is a major public health problem (1). Thus, it is important to investigate how genetic and environmental factors, and their interactions, contribute to colorectal neoplasia. The human cytochrome P450 (CYP) superfamily includes 57 genes encoding enzymes involved in the metabolism of xenobiotics, including drugs and environmental toxins, as well as numerous endogenous compounds (2). CYP2C9 is thought to be involved in the inactivation of 10–30% of clinically useful drugs, the synthesis of important endogenous signaling molecules (i.e., arachidonic acid epoxides), as well as the activation of pro-carcinogenic compounds in tobacco smoke to carcinogenic metabolites (2–7). Notably, common polymorphisms in the CYP2C9 gene modify its enzymatic activity (8–11) and thereby influence response to drugs, such as warfarin (12), and may also modify the effects of endogenous compounds and toxins from cigarette smoke. Previous research suggests that CYP2C9 polymorphisms may be associated with risk of colorectal adenomas or cancer and/or modify the protective effect of aspirin or other non-steroidal anti-inflammatory drugs (NSAIDs) (13–19). In the present study, we investigated associations of two common CYP2C9 polymorphisms with risk of adenoma recurrence and interactions with aspirin treatment and smoking status among participants in a randomized clinical trial of aspirin and folate for the prevention of adenoma recurrence.

Materials and Methods

Study Design and Population

We performed an observational (prospective cohort) analysis of the association between CYP2C9 genotypes and risk of colorectal neoplasia among participants in the Aspirin/Folate Polyp Prevention Study, a placebo-controlled, randomized clinical trial of aspirin and/or folic acid for the prevention of colorectal adenoma recurrence. The parent study design and main findings have been described in detail previously (20, 21). In brief, 1121 participants, who were recruited from 9 clinical centers between 1994 and 1998, had a recent history of one or more histologically confirmed colorectal adenoma and a complete colonoscopy within 3 months prior to enrollment with all known polyps removed from the bowel. Participants agreed to avoid NSAID use during their participation in the study and were randomized to aspirin treatment (placebo, 81 or 325 mg daily) and, independently, to folic acid treatment (placebo or 1 mg daily) in a 3 × 2 factorial design. Per protocol, aspirin treatment was to be continued until a follow-up colonoscopy was performed three years after the baseline colonoscopy. Compliance was excellent. Endpoint data included an assessment of size, number, histology and location of all colorectal lesions detected during randomized aspirin treatment. A single, blinded, study pathologist provided uniform review of all clinical samples removed from the large bowel.

Genotyping

Of the 1048 randomized subjects with adenoma outcome data, 928 were genotyped (88.5%); 120 subjects could not be genotyped due to lack of subject consent or local IRB approval. TaqMan-based assays were performed on two common non-synonymous polymorphisms, CYP2C9*2 (R144C, rs1799853) and CYP2C9*3 (I359L, rs1057910), at the Fred Hutchinson Cancer Research Center using the Applied Biosystems 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). Genotypes were assigned using the Allelic Discrimination Software (Applied Biosystems SDS Software, version 2.3). Primers and cycling conditions are available upon request. For quality control, 7% of the total number of samples were re-genotyped to validate genotype identification protocols. Call rates for all four genotypes exceeded 99% and all were in Hardy-Weinberg equilibrium (p>0.05). Concordance for blinded duplicates was 100%. Laboratory staff were blinded to case-control status for all assays.

Statistical Analysis

The principal outcome was a finding of at least one traditional adenoma at a follow-up colonoscopy after at least one year of randomized treatment and up until the end of randomized aspirin treatment. A secondary “high-risk” outcome was defined as a finding of at least one advanced lesion (a traditional adenoma ≥ 1cm in size, or with villous histology or high grade dysplasia, or cancinoma) and/or multiple adenomas (≥ 3 traditional adenomas). Notably, lesions in the serrated pathway are not included in these outcomes (i.e. serrated or sessile serrated adenomas with or without dysplasia). Risk ratios and 95% confidence intervals used to estimate the association between genotype and outcome were calculated with generalized linear regression models using the Poisson distribution as an approximation to the binomial family and corrected for over or under dispersion. Models were adjusted for age and sex. Genotypes were analyzed using a dominant genetic model due to the limited number of variant homozygotes. For the combined genotype analyses, the variant genotype was defined as having ≥ 1 variant allele of either type (CYP2C9*2 or *3) to maximize power. We also evaluated whether aspirin treatment or smoking status interacted with genotype to modify associations with risk of colorectal neoplasia using multiplicative interaction terms in the regression models and Wald tests. Smoking was defined by self-report as “one cigarette or more each day for a year or more”. Analyses that included aspirin treatment were conducted according to the intention-to-treat principle. Comparisons of selected subject or study characteristics between the groups with and without adenoma recurrence or between groups with wildtype vs. variant genotypes used Pearson χ² tests for categorical variables or two sample t tests for continuous variables. Race/ethnicity was collected by self-report; analyses were performed on all races and on non-Hispanic whites only. All statistical tests were two-sided and considered significant at a value of P<0.05. Stata (version 10) was used for all analyses.

Results

Demographic and other selected characteristics of genotyped participants at enrollment in the Aspirin/Folate Polyp Prevention Study are presented in Table 1 overall (total) and by the primary outcome (adenoma recurrence). Among the 925 genotyped participants, 377 (40.8%) had a recurrence of one or more colorectal adenomas during an average follow-up period of 32.8 months. A higher proportion of participants with adenoma recurrence were current or former smokers (17.1 and 44.5%, respectively) compared to those without adenoma recurrence (12.1 and 41.9%, respectively)(P=0.03). As previously reported for the entire study population (20), individuals in this genotyped subset randomized to treatment with 81 mg/day of aspirin appeared less likely to have adenoma recurrence (30%) compared with those randomized to the placebo arm (35.8%) or to treatment with 325 mg/day aspirin (34.2%)(P=0.09).

Table 1.

Selected characteristics of the study population by adenoma recurrence

Characteristics		Total	No Adenoma	Adenoma Recurrence	P
No. of subjects (%)		925 (100)	548 (9.2)	377 (40.8)
Mean age at ± SD, y enrollment		57.8 ± 9.5	56.6 ± 9.5	59.4 ± 9.2	<0.001
Sex, No. (%)					<0.001
Male		593 (64.1)	322 (58.8)	271 (71.9)
Female		332 (35.9)	226 (41.2)	106 (28.1)
Race/ethnicity. No. (%)					0.21
Non-Hispanic white		799 (86.2)	467 (85.2)	332 (88.1)
Non-Hispanic Black		49 (5.5)	27 (4.9)	22 (5.8)
Hispanic		51 (5.5)	35 (6.4)	16 (4.2)
Other		26 (2.8)	19 (3.5)	7 (1.9)
Smoking status at enrollmenta					0.03
Never		396 (43.0)	252 (46.1)	144 (38.4)
Former		396 (43.0)	229 (41.9)	167 (44.5)
Current		130 (14.0)	66 (12.1)	64 (17.1)
Follow-up time (mean ± SD), mo		32.8 ± 3.8	32.7 ± 3.3	33.0 ± 4.3	0.24
Aspirin treatment group, No. (%)					0.09
Placebo		303 (32.8)	168 (30.7)	135 (35.8)
81 mg/day aspirin		313 (33.8)	200 (36.5)	113 (30.0)
325 mg/day aspirin		309 (33.4)	180 (32.9)	129 (34.2)
Folate treatment group, No. (%)a					0.34
Placebo		419 (49.6)	256 (51.0)	163 (47.7)
1 mg/day folate		425 (50.4)	246 (49.0)	179 (52.3)
CYP2C9*2a	CC	711 (77.0)	429 (78.3)	282 (77.0)	0.30
	TC	200 (21.6)	110 (20.1)	190 (21.6)
	TT	13 (1.4)	9 (1.6)	4 (1.4)
CYP2C9*3	AA	824 (89.1)	504 (92.0)	320 (84.9)	0.002
	AC	100 (10.8)	43 (7.8)	57 (15.1)
	CC	1 (0.1)	1 (0.2)	0
Combined genotypeb	CCc, AA	626 (67.8)	394 (71.9)	232 (61.7)	0.001
	TC, TT or AC, CCd	298 (32.3)	154 (28.1)	144 (38.3)

CYP2C9*2 = rs1799853, 430 C>T, R144C; CYP2C9*3 = rs1057910, 1075 A>C, I359L.

^aSmoking status is missing for 3 subjects; 81 subjects were not randomized to folate treatment; CYP2C9*2 genotype is missing for 1 subject.

^bFor the combined genotype analysis, the variant genotype was defined as having ≥ 1 variant allele of either type (CYP2C9*2 or *3).

^cHere “CC” is CYP2C9*2 homozygous wildtype genotype.

^dHere “CC” is CYP2C9*3 homozygous variant genotype.

Overall, among all genotyped participants, 213 (23%) had one or more CYP2C9*2 variant alleles (minor allele frequency (MAF)=12.2%), 101 (10.9%) had one or more CYP2C9*3 variant alleles (MAF=5.5%) and 298 participants (32.3%) had one or more variant allele of either type (combined genotype) (Table 1). Notably, a higher proportion of individuals with adenoma recurrence had a CYP2C9*3 variant genotype (15.1%) compared to those without adenoma recurrence (8%) (P=0.002), whereas the proportion of CYP2C9*2 variant genotype was similar between the two groups (P=0.30). When the CYP2C9 combined genotype was examined, a higher proportion of individuals with adenoma recurrence had one or more variant alleles (38.3%) compared to those without adenoma recurrence (28.1%) (P=0.001).

As shown in Table 2, most subject and study characteristics did not differ by CYP2C9 genotype, including age, sex, aspirin or folate treatment groups and mean follow-up time. However, the CYP2C9*2 genotype was associated with race/ethnicity: a higher proportion of individuals with one or more variant alleles were non-Hispanic white (94.4%) compared to the proportion of wild type individuals that were non-Hispanic white (84%) (P=0.001). In addition, the CYP2C9*3 genotype appeared to be associated with smoking status, such that a higher proportion of individuals with one or more variant alleles were former (45.5%) or current smokers (20.8%) compared to the proportion of wild type individuals that were former (42.6%) or current smokers (13.3%)(P=0.05).

Table 2.

Selected characteristics of the study population by CYP2C9 genotypes

Characteristics:	CYP2C9*2a			CYP2C9*3
	CC	TC/TT	P	AA	AC/CC	P
Mean age, ± SD, y	57.6 ± 9.5	58.2 ± 9.3	0.42	57.8 ± 9.5	57.4 ± 9.6	0.71
Sex, No. (%)			0.59			0.70
Male	453 (63.7)	140 (65.7)		530 (64.3)	63 (62.4)
Female	258 (36.3)	73 (34.3)		294 (35.7)	38 (37.6)
Race/ethnicity. No. (%)			0.001			0.42
Non-Hispanic white	597 (84.0)	201 (94.4)		708 (85.9)	91 (90.1)
Non-Hispanic Black	43 (6.0)	6 (2.8)		47 (5.7)	2 (2.0)
Hispanic	45 (6.3)	6 (2.8)		45 (5.5)	6 (5.9)
Other	26 (3.7)	0 (0)		24 (2.9)	2 (2.0)
Smoking statusa			0.16			0.05
Never	304 (42.9)	92 (43.2)		362 (44.1)	34 (33.7)
Former	296 (41.8)	99 (46.5)		350 (42.6)	46 (45.5)
Current	108 (15.3)	22 (10.3)		109 (13.3)	21 (20.8)
Follow-up time (mean ± SD), mo	32.8 ± 3.6	32.9 ± 4.3	0.91	32.8 ± 3.7	33.1 ± 3.8	0.38
Aspirin group, No. (%)			0.57			0.76
Placebo	238 (33.5)	65 (30.5)		271 (32.9)	32 (31.7)
81 mg/day aspirin	242 (34.0)	71 (33.3)		281 (34.1)	32 (31.7)
325 mg/day aspirin	231 (32.5)	77 (36.2)		272 (33.0)	37 (36.3)
Folate group, No. (%)a			0.78			0.68
Placebo	327 (49.9)	91 (48.7)		373 (49.4)	46 (51.7)
1 mg/day folate	329 (50.1)	96 (51.3)		382 (50.6)	43 (48.3)

CYP2C9*2 = rs1799853, 430 C>T, R144C; CYP2C9*3 = rs1057910, 1075 A>C, I359L.

^aSmoking status is missing for 3 subjects; 81 subjects were not randomized to folate treatment; CYP2C9*2 genotype is missing for 1 subject.

We examined the association of CYP2C9 genotypes with risk of colorectal neoplasia during the period of randomized aspirin treatment by assessing the occurrence of any adenoma or of advanced lesions/multiple adenomas (Table 3). The combined variant genotype (one or more variant allele of either type) was associated with statistically significant increased risks of 29% for any adenoma (RR=1.29, 95% CI=1.09–1.51, P=0.002) and 64% for advanced lesions or multiple adenomas (RR=1.64; 95% CI=1.18–2.28, P=0.003). The increase in risk was mostly due to the CYP2C9*3 genotype: having one or more variant allele was associated with increased risks of 47% (RR=1.47, 95% CI=1.19–1.83, P<0.001) for any adenoma and 79% (RR=1.79, 95% CI=1.16–2.75, P=0.008) for advanced lesions or multiple adenomas. However, the CYP2C9*2 variant was also associated with non-statistically significant increased risks, especially for the “high-risk” findings (advanced lesions or multiple adenomas). As shown in Table 3, very similar results were observed when the analyses were restricted to non-Hispanic whites (86.2% of the study population).

Table 3.

Risk of any adenoma recurrence (top) or advanced lesions/multiple adenomas (bottom) by CYP2C9 genotypes

Outcome	Subjects	Genotypeb	# with outcome / # totalc	RR (95% CI)d	Pe
Any adenoma	All races	Variant (combined)	144/298	1.29 (1.09–1.51)	0.002
		CYP2C9*2	94/213	1.09 (0.91–1.31)	0.33
		CYP2C9*3	57/101	1.47 (1.19–1.83)	<0.001
	Non-Hispanic whites	Variant (combined)	135/277	1.27 (1.07–1.50)	0.005
		CYP2C9*2	90/201	1.09 (0.90–1.31)	0.37
		CYP2C9*3	51/91	1.43 (1.13–1.79)	0.002
Advanced lesions or multiple adenomasa	All races	Variant (combined)	55/295	1.64 (1.18–2.28)	0.003
		CYP2C9*2	35/210	1.29 (0.89–1.85)	0.18
		CYP2C9*3	22/100	1.79 (1.16–2.75)	0.008
	Non-Hispanic whites	Variant (combined)	53/274	1.84 (1.29–2.62)	0.001
		CYP2C9*2	34/198	1.38 (0.94–2.01)	0.10
		CYP2C9*3	21/90	1.94 (1.24–3.03)	0.003

^aAn outcome of advanced lesions (adenomas ≥1cm, or with villous histology or high grade dysplasia, or arcinoma) or multiple adenomas (≥3); data is missing for two subjects with unknown adenoma size.

^bFor combined genotype analysis, variant (combined) = one or more variant (minor) allele at either CYP2C9*2 (rs1799853, R144C) or CYP2C9*3 (rs1057910, I359L). For single SNP analyses, both SNPs are coded with a dominant genetic model.

^cThis is the # of subjects with ≥1 variant allele with the specified outcome/total # of subjects with ≥1 variant allele.

^dRelative risk (RR) adjusted for sex and age for comparisons with homozygous wild type; 95% confidence interval (95% CI).

^eWald test for the comparison to wild-type genotype.

We also investigated whether CYP2C9 genotype modified the effect of aspirin treatment or the association of smoking status with risk of any adenoma (Table 4). Among all genotyped participants, the lower dose of aspirin treatment (81 mg) reduced risk by 19% (RR=0.81, 95% CI=0.67–0.98, P=0.03) whereas the higher dose (325 mg) appeared ineffective (RR=0.94 RR, 95% CI=0.78–1.13, P=0.48) (not shown). When aspirin treatment was stratified by CYP2C9 genotype (Table 4), similar results were obtained regardless of genotype and there was no evidence for an interaction of genotype and aspirin treatment. Notably, the results were similar when this analysis was restricted to non-Hispanic whites (Table 4) or to the CYP2C9*3 genotype (not shown). In addition, CYP2C9 genotype also did not modify the effect of folate treatment (not shown).

Table 4.

Interaction of CYP2C9 genotype and aspirin treatment or smoking status on risk of any adenoma recurrence

Subjects	Genotype	RR (95% CI)b			Pintc
		Aspirin Treatment Group
		Placebo	81 mg/day	325 mg/day
All races	Wild-type	1.0 (referent) [86/210]	0.79 (0.61–1.02) [68/214]	0.95 (0.74–1.21) [78/202]	0.85
	Varianta	1.0 (referent)d [49/93]	0.81 (0.60–1.08) [45/99]	0.87 (0.65–1.16) [50/106]
Non-Hispanic Whites	Wild-type	1.0 (referent) [71/170]	0.81 (0.61–1.07) [58/178]	0.94 (0.72–1.22) [67/173]	0.97
	Varianta	1.0 (referent)e [44/85]	0.83 (0.61–1.12) [44/96]	0.91 (0.69–1.22) [47/96]
		Smoking Status
		Never	Former	Current
All races	Wild-type	1.0 (referent) [82/274]	1.26 (0.99–1.58) [105/258]	1.60 (1.19–2.15) [41/91]	0.04
	Varianta	1.0 (referent)f [62/122]	0.80 (0.62–1.03) [61/137]	1.03 (0.72–1.50) [21/39]
Non-Hispanic Whites	Wild-type	1.0 (referent) [72/233]	1.21 (0.95–1.56) [92/218]	1.47 (1.05–2.08) [30/67]	0.04
	Varianta	1.0 (referent)g [62/118]	0.76 (0.58–0.99) [55/124]	0.98 (0.67–1.43) [18/35]

^aCombined genotype analysis where variant = one or more variant (minor) alleles at either CYP2C9*2 (R144C, rs1799853) or CYP2C9*3 (I359L, rs1057910).

^bRelative risk (RR) adjusted for age and sex; 95% confidence interval (95% CI); [#/#] = [# subjects with adenoma/total # subjects]

^cP_int = P for interaction of genotype with aspirin treatment or smoking status.

^dRR (95% CI) for variant vs. wild-type is 1.31 (1.00–1.72) among placebo subjects.

^eRR (95% CI) for variant vs. wild-type is 1.28 (0.96–1.71) among placebo subjects.

^fRR (95% CI) for variant vs. wild-type is 1.67 (1.30–2.16) among never smokers.

^gRR (95%CI) for variant vs. wild-type is 1.65 (1.27–2.14) among never smokers.

On the other hand, there was evidence for an interaction between smoking status and CYP2C9 genotype (Table 4). Among participants with the wild-type CYP2C9 genotype, smoking was associated with increased adenoma risks of 26% (RR=1.26, 95% CI=0.99–1.58) for former smokers and 60% (RR=1.60, 95% CI=1.19–2.15) for current smokers. However, in participants with one or more variant alleles of either type, smoking status was not associated with increased risks: RR=0.80 (95% CI=0.62–1.03) in former smokers and RR=1.03 (95% CI=0.72–1.50) in current smokers. In this stratified analysis, the combined CYP2C9 variant genotype (one or more variant allele of either type) was associated with a 67% increased risk of adenoma among non-smokers (RR=1.67, 95% CI= 1.30–2.16, Table 4 footnote) compared to the 29% increased risk seen in all subjects (Table 3). Similar results were seen when the analyses were restricted to non-Hispanic whites. The interaction between smoking status and CYP2C9 genotype was statistically significant for all participants (P=0.04) as well as for non-Hispanic whites (P=0.04). When this analysis was restricted to the CYP2C9*3 genotype, similar results were seen, although the interaction was not statistically significant (not shown).

Discussion

In this prospective analysis of participants from a randomized clinical trial of aspirin for colorectal adenoma chemoprevention, CYP2C9 genotype was associated with increased risks of any adenoma of 29% for ≥1 variant of either type (CYP2C9*2 or *3) and 47% for ≥1 CYP2C9*3 variant. Moreover, the associations with “high-risk” findings (multiple adenomas (≥3) and/or advanced lesions), appeared larger: risks increased by 64% for ≥1 variant of either type (CYP2C9*2 or *3) and 79% ≥1 CYP2C9*3 variant. In addition, although CYP2C9 genotype didn’t modify the protective effect of aspirin on adenoma risk, it substantially modified the association with smoking such that former or current smoking was only associated with increased risk of adenoma among participants with the wild-type CYP2C9 genotype.

There is evidence from recent tissue microarray experiments that the CYP2C9 enzyme is broadly expressed in human tissues, including moderate expression in the colon (22), and is the most abundantly expressed epoxygenase in several human malignant neoplasms (23). The stronger association of CYP2C9 genotype with “high-risk” findings (advanced and multiple adenomas) in the current study is noteworthy because of the potential clinical importance of these lesions, given their association with greater risk for future advanced neoplasia and cancer (24). Importantly, we observed similar results when our analyses were restricted to non-Hispanic whites, mitigating concerns about potential effects of population stratification. In addition, the reduction in enzyme activity for the CYP2C9*3 variant is more substantial than for the CYP2C9*2 variant (8–10), so the stronger associations observed with the CYP2C9*3 variant genotype in the current work is consistent with a role for this enzyme in modulating adenoma risk. However, others have suggested that associations with the CYP2C9*2 genotype may actually be due to linkage disequilibrium with the CYP2C8*3 variant rather than a true effect of the CYP2C9 polymorphism (see (11, 17, 25, 26)).

A number of previous studies have investigated associations of these CYP2C9 polymorphisms with colorectal adenomas and cancer. Among previous studies of CYP2C9 genotype and adenoma risk, Chan et al. also reported a statistically significant increase in risk of distal adenoma associated with combined (CYP2C9*2 or *3) variant genotype in the Nurses Health Study (14). However, there were only modest, non-statistically significant increased risks in several other studies (13, 15, 17) and one report of decreased risk in a small Scottish case-control study (19).

Among these prior studies, one reported that CYP2C9 genotype modified the protective association with aspirin, although it was not clear if this interaction was statistically significant (13), whereas two others noted no interaction (14, 15). Our trial, with its larger size and randomized aspirin intervention, was well suited to address this issue, which was a major impetus for the current work (27, 28). However, since the CYP2C9 enzyme is thought to have a relatively minor role in metabolizing aspirin (10), it is perhaps not surprising that we did not find an interaction. Only one of the previously mentioned studies investigated the interaction between genotype and smoking on adenoma prevalence, and no association was found (14). Of studies investigating associations with colorectal cancer risk, the two largest reported either no association (16) or a decreased risk associated with the CYP2C9*2 genotype (18), and no interaction with aspirin (16) or smoking (18).

The apparent association of CYP2C9*3 genotype with smoking status in our study population may be a chance finding or may reflect a previously unrecognized role for this enzyme in metabolizing nicotine. Although a large genome wide association study meta-analysis did not identify genetic variation in the vicinity of this gene to be associated with smoking behaviors (29), it is possible that there were effects that were not detected at a genome wide significance level. Interestingly, the CYP2C9 gene is located on chromosome 10q24 and a marker in the adjacent 10q25 region was associated with smoking behavior in a study of twins from Finland (30). In addition, another member of the CYP2 gene family, CYP2A6, has been associated with smoking behaviors and lung cancer risk (31).

The mechanism by which CYP2C9 genotype may increase adenoma risk is not known. However, the CYP2C9 enzyme may play a role in the metabolism of arachidonic acid to eicosanoids, specifically to epoxyeicosatrienoic acid, and may ultimately influence carcinogenesis through effects on cellular proliferation and inflammation (2, 3, 8). In addition to effects on endogenous modulators, CYP2C9 may be involved in the metabolism of environmental carcinogens impacting colorectal carcinogenesis. The attenuated risk associated with cigarette smoking among participants with the variant genotype in the current study could be explained by a role for CYP2C9 in activation of procarcinogenic chemicals such as polycyclic aromatic hydrocarbons found in tobacco smoke (4–7, 32) (33). The increased adenoma risk associated with smoking observed in our population is consistent with the results of published meta-analyses for colorectal adenoma (34) and cancer (35, 36). Since smoking appears to increase risk of certain histologically and molecularly defined subsets of colorectal cancer (37, 38)), which can also result in differential interactions (see(39)), it will be important to explore associations with CYP2C9 genotype among these subsets in future studies. Likewise, it seems possible that differences in the types of pre-cancerous lesions examined and/or smoking status among different study populations may explain some of the heterogeneity observed in published studies of associations between CYP2C9 genotype and adenoma risk and this will be an important avenue for future exploration.