Polymorphisms in PTGS1, PTGS2 and IL-10 do not influence colorectal adenoma recurrence in the context of a randomized aspirin intervention trial
Regular use of aspirin and other nonsteroidal antiinflammatory drugs reduces both the development of colorectal neoplasia and recurrence of colorectal adenoma (CRA). Modulation of the effects of aspirin by genetic factors has been reported, potentially allowing targeting of treatment to individuals most likely to gain benefit. Prostaglandin H synthase 1 (PTGS1) and PTGS2 are key enzymes in prostaglandin synthesis and are inhibited by aspirin, whilst interleukin-10 (IL-10) is an important antiinflammatory cytokine. We investigated whether functional genetic polymorphisms in the PTGS1, PTGS2 and IL-10 genes influence CRA recurrence in individuals participating in a randomized aspirin intervention trial. DNA was available for genotyping from 546 patients who received aspirin (300 mg daily) or placebo for a mean 41-months' duration. Homozygote carriers of variant alleles for the PTGS1 50C>T, PTGS2 −765G>C and IL-10 −592C>A polymorphisms did not have a significantly altered risk of CRA recurrence (relative risk [RR] = 0.91; 95% confidence interval [CI]: 0.14–6.07, RR = 1.32; 95%CI: 0.66–2.62 and RR = 1.24; 95% CI: 0.74–2.07, respectively). There were also no significant interactions between aspirin intervention and genotype in determining recurrence risk. These data indicate that these polymorphisms are unlikely to influence CRA recurrence and cannot be used to identify individuals who derive benefit from aspirin intervention. © 2007 Wiley-Liss, Inc.
Regular use of aspirin or nonsteroidal antiinflammatory drugs (NSAIDs) has consistently been reported to result in an ∼50% reduction in incidence of colorectal cancer (CRC), and 3 randomized intervention trials have demonstrated a reduction in colorectal adenoma (CRA) recurrence in patients receiving aspirin.1, 2, 3, 4 A benefit in preventing colorectal neoplasia is not, however, observed in all patients using aspirin, and it is very likely that other environmental exposures and genetic variation play a part in determining an individuals' response.
The enzymes prostaglandin H synthase 1 (PTGS1, or cyclooxygenase1 (COX1)) and PTGS2 (COX2) catalyze prostaglandin synthesis, and play important roles in regulating both constitutive and reactive inflammation.5 Both enzymes are inhibited by aspirin and other NSAIDs, and this inhibition is thought to mediate at least part of the protective effect of these drugs in colorectal carcinogenesis.6 Interleukin-10 (IL-10) is an antiinflammatory cytokine that inhibits synthesis of the proinflammatory cytokines IL-1β, IL-6, IL-8 and IL-2, and has a crucial role in modulating gastrointestinal tract inflammation which is implicated in colorectal carcinogenesis.7, 8, 9 Polymorphisms within the promoter or coding regions of the PTGS1, PTGS2 and IL-10 genes have been documented to alter protein expression, and in case–control studies have been reported to influence risk of developing colorectal neoplasia.10, 11, 12, 13, 14, 15 Furthermore, the PTGS1 50C>T (rs3842787), PTGS2 −765G>C (rs20417) and IL-10 −592C>A (rs 1800872) polymorphisms have been demonstrated to interact with aspirin or NSAID use in determining CRC or CRA risk, with the benefits of these drugs being confined to individuals with certain genotypes.13, 15, 16
We sought to further investigate the role of these polymorphisms in colorectal neoplasia by genotyping patients participating in a randomized intervention trial of aspirin for the prevention of CRA recurrence.
Material and methods
The United Kingdom Colorectal Adenoma Prevention (UKCAP) trial is a recently completed multicenter randomized placebo-controlled intervention trial for the prevention of CRA recurrence.3 Eligible subjects were recruited from 1997 to 2001, had ≥1 histologically confirmed CRA, ≥0.5 cm in size, detected at full colonoscopic examination and were not already taking regular aspirin, nonaspirin NSAID or prescribed folate supplements. Patients were randomized to aspirin alone (300 mg daily), folate alone (500 μg daily), both aspirin and folate, or double placebo. The primary endpoint was histologically confirmed recurrence of CRA or CRC. Recurrence was ascertained at follow-up colonoscopy scheduled for 3 years after entry colonoscopy, or performed earlier if symptoms dictated (mean 41 months). If follow-up colonoscopy was performed prior to the 3-year time-point and CRA or CRC was found, then the patient left the trial; if no adenoma was found then the patient continued on trial medication and underwent further colonoscopy at the 3-year time-point. Histopathology was performed at local hospitals without central review. Suspected recurrences found at follow-up colonoscopy were reviewed by the same histopathology department as the original trial entry specimen. Compliance with trial medication was assessed at 4-monthly intervals, and there was ≥85% compliance with prescribed tablets in the aspirin arms of the trial.
Follow-up colonoscopy was performed on 853 patients, and DNA was available from 546 patients all of whom were of Caucasian ethnicity. Not all patients from the original trial could be included in this molecular subprotocol as some could not be contacted, and others did not consent to DNA analysis. Informed consent for the study was obtained from all participants and the study was carried out with ethical review board approval in accordance with the tenets of the declaration of Helsinki.
PTGS1 50C>T, PTGS2 −765G>C and IL-10 −592C>A genotypes were generated from germline DNA using Taqman technology implemented on an ABI 7900HT sequence detection system (Applied Biosystems, Foster City, USA). Genotyping assays for each polymorphism were validated using control samples of known homozygote wild-type, heterozygote and homozygote variant genotype generated by direct sequencing. Unblinded control samples were included on each sample plate, and were correctly genotyped on 100% of occasions. Laboratory personnel employed in genotyping patient DNAs were blinded to clinical outcome.
Baseline characteristics between the total UKCAP trial population and the genotyped subgroup were compared using the χ2 and t-tests. Genotype frequencies were tested for departure from Hardy–Weinberg equilibrium (HWE) using the χ2 test. The presence or absence of colorectal neoplasia recurrence was considered a binary outcome in the cohort study. The relationship between genotype and risk of colorectal neoplasia recurrence was assessed by means of relative risks (RRs) and 95% confidence intervals (CIs) calculated using Poisson regression with robust error variance, adjusting for sex and interval between entry and follow-up colonoscopy, since these two variables were found to significantly influence recurrence risk.17, 18 For each polymorphism, homozygous wild-type patients were compared to heterozygous and homozygous variant patients separately and combined. The likelihood ratio test was used to explore interactions between genotype and aspirin treatment with respect to recurrence risk by comparing models with and without a multiplicative term for the 2 variables. For the interaction analyses, patients were divided into aspirin-treated (aspirin alone, and aspirin and folate intervention groups), and nonaspirin-treated (folate alone, and double-placebo intervention groups) on an intention-to-treat basis. To maximize power in the interaction analyses, patients with heterozygote and homozygous variant genotypes were combined and compared to homozygous wild-type patients.
Statistical analyses were undertaken using STATA, version 7.0 (Stata Corporation, College Station, TX). All tests were 2-sided, and a p value less than 0.05 was considered significant.
There were no significant differences in age, sex, intervention group, interval between entry and follow-up colonoscopy, and outcomes between the total UKCAP trial population and patients included in the genotyping analysis (Table I). Of the 546 patients included in the genotyping analysis, 130 (23.8%) had ≥1 CRA and 7 (1.3%) CRC detected at follow-up colonoscopy. Seventy patients (12.8%) had advanced colorectal neoplasia, defined as CRAs with villous or tubulovillous features, size ≥1 cm, severe dysplasia or invasive carcinoma. In the main trial a reduced CRA recurrence risk was observed in patients who received aspirin (RR = 0.81; 95% CI: 0.66–1.02),3 and in the genotyped subgroup a reduced recurrence risk was also seen (RR = 0.92; 95% CI: 0.70–1.22).
Table I. Comparison of the Total UKCAP Trial Population and Patients Genotyped in this Study
|Age1|| || |
| Mean (yrs)||57.5||57.3|
| Std. dev.||9.3||9.3|
|Sex|| || |
| Male||477 (56)2||289 (53)|
| Female||376 (44)||256 (47)|
|Intervention|| || |
| Folate alone||215 (25.2)||144 (26.4)|
| Aspirin alone||217 (25.4)||131 (24.0)|
| Folate and aspirin||217 (25.4)||135 (24.7)|
| Double placebo||204 (23.9)||136 (24.9)|
|Colonoscopy interval3|| || |
| Mean (months)||40.3||40.7|
| Std. dev. (months)||12.2||11.7|
| Range (months)||2–79||6–74|
|Outcome4|| || |
| Adenoma||207 (24.2)||130 (23.8)|
| Carcinoma||11 (1.3)||7 (1.3)|
| Advanced neoplasia||104 (12.2)||70 (12.8)|
Allele frequencies for the PTGS1 50T, PTGS2 −765C and IL-10 −592A alleles were 7, 14 and 26%, respectively, consistent with previous reports in Caucasian populations, and genotype frequencies for all 3 polymorphisms were in HWE.15, 16, 19 When risk of colorectal neoplasia recurrence was stratified by PTGS1 50C>T, PTGS2 −765G>C or IL-10 −592C>A genotypes, no statistically significant associations were observed (Table II). Similarly, when the analysis was restricted to advanced lesions there were no significant influences of genotype on recurrence risk (Table II).
Table II. Risk of Detection of any Colorectal Neoplasia and Advanced Neoplasia at Follow-Up Colonoscopy Stratified by Genotype
|50C>T||CT||15/46||1.01 (0.66–1.54)3||6/55||0.75 (0.36–1.58)|
| ||TT||1/4||0.91 (0.14–6.07)||0/5||–|
| ||CT/TT||16/50||1.00 (0.66–1.52)||6/60||0.70 (0.33–1.48)|
|−765G>C||GC||33/91||0.96 (0.70–1.30)||16/108||0.86 (0.52–1.43)|
| ||CC||5/7||1.32 (0.66–2.62)||3/9||1.49 (0.62–3.57)|
| ||GC/CC||38/98||0.99 (0.74–1.33)||19/117||0.92 (0.57–1.47)|
|−592C>A||CA||51/150||1.11 (0.83–1.47)||25/176||1.02 (0.66–1.58)|
| ||AA||11/28||1.24 (0.74–2.07)||4/35||0.84 (0.32–2.22)|
| ||CA/AA||62/178||1.13 (0.86–1.48)||29/211||0.99 (0.65–1.51)|
Risks of colorectal neoplasia recurrence following stratification by both genotype and aspirin intervention are detailed in Table III. There were no statistically significant interactions between genotype and aspirin intervention for any polymorphism. Nor were there significant interactions when the analysis was restricted to advanced lesion recurrence (data not shown).
Table III. Risk of Detection of Colorectal Neoplasia Stratified by Genotype and Intervention
|50C>T||CT/TT||8/20||0.97 (0.55–1.72)2||8/30||0.95 (0.51–1.78)|
| || || ||p interaction = 0.91|| || |
|−765G>C||GC/CC||19/49||0.87 (0.58–1.31)||19/49||0.97 (0.65–1.45)|
| || || ||p interaction = 0.73|| || |
|−592C>A||CA/AA||34/93||1.07 (0.74–1.53)||28/85||1.06 (0.72–1.58)|
| || || ||p interaction = 0.74|| || |
Our study is the first to report on the influence of polymorphisms in the PTGS1 and PTGS2 genes and risk of CRA recurrence, and also the first to investigate the IL-10 −592C>A polymorphism in this setting. The variant PTGS1 50T allele results in an amino acid change in the signal peptide of the PTGS1 protein, and significantly increased inhibition of the PTGS1 enzyme by aspirin has been reported in heterozygote carriers of the variant −50T allele.10 The PTGS2 −765G>C polymorphism is located within a putative binding site for stimulatory protein 1, which is considered to be a positive activator for PTGS2 expression.11 Heterozygous or homozygous carriers of the variant PTGS2 −765C allele have been reported to show significantly less PTGS2 expression in normal gastrointestinal mucosa, and a significantly reduced increase in PTGS2 expression following surgery.20, 21 The IL-10 −592C>A polymorphism is in complete linkage disequilibrium (LD) with a second IL-10 promoter variant, −819C>T and together with a third variant −1082G>A forms 3 haplotypes (GCC, ACC and ATA).15 The ATA haplotype has been reported to be associated with reduced IL-10 expression compared to the GCC haplotype.12, 22 Thus there is evidence that all 3 polymorphisms investigated in this study are functional with significant influences on protein expression.
Although a case–control study reported no influence of PTGS1 50C>T genotype on risk of developing CRA, homozygote carriers of the PTGS1 50C allele showed a reduced CRA risk if they reported regular aspirin or NSAID use, whilst individuals with 1 or 2 PTGS1 50T alleles showed no benefit from NSAID use.13 This finding, however, is seemingly at odds with the in vitro findings of increased aspirin inhibition of the PTGS1 enzyme in PTGS1 50T carriers. Similarly, in the same group of CRA cases and control subjects, no direct influence of PTGS2 −765G>C genotype on risk of developing CRA was reported, but a significant interaction between genotype and aspirin or NSAID use was noted, with the benefits of NSAIDs being confined to carriers of PTGS2 −765G alleles.16 No such interaction was observed in a subsequent study of PTGS2 −765G>C genotype and CRA risk, although this was of smaller sample size.23 Two case–control studies investigating the PTGS2 −765G>C polymorphism in the context of CRC risk also reported no direct influence of genotype, but no data on the interaction between genotype and aspirin or NSAID use in determining CRC risk was presented.14, 24 In a Scottish population the IL-10 −592C>A polymorphism was reported to interact with aspirin use in predicting risk of CRC development, with the benefits of aspirin being confined to −592A allele carriers, although a similar interaction was not observed with nonaspirin NSAID use.15 No direct association of IL-10 −592C>A genotype and CRC risk was observed.
In our study, no direct influences on risk of CRA recurrence were conferred by PTGS1 50C>T, PTGS2 −765G>C or IL-10 −592C>A genotypes, and these findings would appear to be consistent with the previous epidemiological studies reporting on the relationship between genotype and colorectal neoplasia risk. However, in our study there were also no significant interactions between aspirin intervention and genotype in determining CRA recurrence risk, as was reported in some case–control studies. These differences in results may reflect genuine differences in the genotype-drug interactions at different stages of colorectal carcinogenesis (adenoma development as opposed to adenoma recurrence), differences in drug exposures (both dose and duration) or may be due to sample size or chance. In our study, patients randomized to aspirin treatment were exposed to a relatively high dose of aspirin, 300 mg daily, and there was also a high level of compliance with aspirin treatment. This dose and frequency is higher than that typically employed in case–control studies to define individuals as “aspirin-exposed,” and would be expected to maximize the likelihood of observing any genotype–drug interaction.15, 16 It is also possible, however, that such interactions require a longer duration of exposure to exert their influence on colorectal carcinogenesis than the mean 41-month period in this study. Although aspirin treatment reduced CRA recurrence in the main UKCAP trial, in the genotyped subgroup, the protective effect was more limited. This will have reduced the power of our study to detect an interaction between aspirin and genotype, and could also account for the absence of such interactions in our study. While our study had 80% power to detect the main effect of genotype on recurrence risk (assuming a variant allele frequency of 0.3 and RR of recurrence of 0.5 associated with presence of the variant allele in a dominant model), power to detect interactions between genotype and aspirin treatment in determining recurrence risk was limited (32% assuming a RR of recurrence of 0.92 associated with aspirin treatment as observed in this study).
A previous study has reported on IL-10 −819C>T and −1082G>A polymorphism genotype and CRA recurrence risk in participants in a dietary intervention trial.25 In this study, neither variant influenced recurrence risk directly, and there was no significant interaction between the IL-10 −819C>T and NSAID use in determining recurrence risk. Since the IL-10 −819C>T and −592C>A variants are in strong LD, these results corroborate the findings of our study. A significant interaction between IL-10 −1082G>A genotype and NSAID use was reported in the study by Sansbury et al.,25 which was not observed in the case–control study by Macarthur et al.,15 highlighting the differences in genotype–drug interactions that may occur at different stages of colorectal carcinogenesis.
In summary, our results indicate that the PTGS1 50C>T, PTGS2 −765G>C and IL-10 −592C>A polymorphism genotypes are unlikely to influence CRA recurrence, or interact with aspirin in determining CRA recurrence risk. As such in patients diagnosed with CRA, these polymorphisms are unlikely to be clinically useful in identifying those who will gain differential benefit from aspirin intervention in preventing recurrence.
R.A.H. is in receipt of a Cancer Research UK Clinical Research Training Fellowship.