1 Nutrition and Toxicology Research Institute Maastricht, Department of Epidemiology, Universiteit Maastricht, Maastricht, the Netherlands
2 Division of Human Nutrition, Wageningen University and Research Centre, Wageningen, the Netherlands
3 Department of Pathology, University Medical Centre St. Radboud, Nijmegen, the Netherlands
4 Nutrition and Toxicology Research Institute Maastricht, Department of Pathology, Universiteit Maastricht, Maastricht, the Netherlands
5 TNO Nutrition and Food Research, Zeist, the Netherlands
6 Research Institute Growth and Development, Department of Pathology, Universiteit Maastricht, Maastricht, the Netherlands
Correspondence to Dr. M. P. Weijenberg, Department of Epidemiology, Universiteit Maastricht, P.O. Box 616, 6200 MD Maastricht, the Netherlands (e-mail: mp.weijenberg{at}epid.unimaas.nl).
Received for publication September 23, 2004. Accepted for publication December 22, 2004.
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ABSTRACT |
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adenomatous polyposis coli; colorectal neoplasms; glutathione transferase; neoplasm proteins; polymorphism, genetic; smoking
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INTRODUCTION |
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If cigarette smoking is an early event in tumorigenesis, long-term follow-up should show a positive association between smoking and colorectal cancer when the extensive lag time (over 30 years) for the carcinogenic process is taken into account (3). Indeed, Giovannucci et al. (4
, 5
) found a twofold increase in risk of colorectal cancer after 35 years of smoking. This finding was supported by a report from another long-term US cohort study (6
) but not by other reports (7
9
).
Most sporadic colorectal cancers are thought to arise through underlying genetic pathways, which involve aberrations in a number of genes, as described by Fearon and Vogelstein (10). Inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene is thought to be a key event early in colorectal tumorigenesis. This is underscored by findings of somatic mutations in the mutation cluster region of APC, leading to a truncated and therefore inactive APC protein in the majority of sporadic colorectal adenomas and carcinomas (11
13
). We previously reported on the occurrence of a large number of mutations in the mutation cluster region of the APC gene; however, these would lead to a truncated APC protein in just 37 percent of patients (14
). Because smoking is thought to be involved in the early stages of colorectal tumorigenesis, smoking may be associated with mutations in the APC gene. This is supported by results from rodent studies in which benzo(a)pyrene was found to induce G
T transversions and N-nitrosamines were found to induce G:C
A:T transitions in ras oncogenes (15
), both of which are important constituents of cigarette smoke.
Colorectal cancer can be divided broadly into two subgroups based on genomic instability. Whereas the majority of sporadic colorectal tumors are chromosomally unstable, a smaller subset (1015 percent) exhibit loss of mismatch repair as manifested by microsatellite instability. In approximately 90 percent of microsatellite-unstable tumors, absence of human mut-L homologue 1 (hMLH1) expression was observed (16
). Studies have shown that microsatellite instability and mutations in the APC, TP53, and K-ras genes occur almost mutually exclusively (17
19
), which suggests that these aberrations represent separate pathways. The risk of smoking may be associated with the specific subset of colorectal tumors displaying microsatellite instability. Cigarette smoking was found to be associated with microsatellite-unstable colorectal tumors in three case-control studies (20
22
), whereas another case-control study could not confirm this (23
).
Persons with a homozygous deletion of the glutathione-S-transferase M1 (GSTM1) gene or the glutathione S-transferase T1 (GSTT1) gene have no enzyme expression and may therefore be at an increased risk of developing colorectal cancer when exposed to carcinogens from cigarette smoke. However, previous reports on the interaction between tobacco smoking and GSTM1 and/or GSTT1 have not shown a relation between smoking and genotype (2428
). In the absence of data on the GSTM1 and GSTT1 genotypes in controls, a case-only design provides an efficient means of estimating possible interactions with a greater precision than can be obtained from case-control studies (29
).
Our aim in this analysis was to investigate associations between cigarette smoking and overall colorectal cancer risk in a prospective cohort study. In addition, we studied associations between smoking and (specific) mutations in the APC gene and absence of hMLH1 expression, which are thought to represent distinct mechanisms involved in colorectal carcinogenesis. We evaluated possible interactions between cigarette smoking and the GSTM1 and GSTT1 genotypes in a nested case-only design.
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MATERIALS AND METHODS |
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Incident cancer cases are identified by monitoring of the entire cohort for cancer occurrence through annual record linkage to the Netherlands Cancer Registry (nine regional cancer registries throughout the Netherlands) and the Pathologisch Anatomisch Landelijk Geautomatiseerd Archief (PALGA), a nationwide network and registry of histo- and cytopathology (31). Together, the Netherlands Cancer Registry and PALGA provide nearly 100 percent coverage of the municipalities included in the Netherlands Cohort Study on Diet and Cancer.
Accumulation of person-time in the cohort was estimated through biennial vital status follow-up of a subcohort of 3,500 men and women who were randomly selected after baseline exposure measurement (31). Cases with prevalent cancer other than nonmelanoma skin cancer were excluded from the subcohort, resulting in a subcohort of 3,346 men and women. No subcohort members were lost to follow-up.
The first 2.3 years of follow-up were excluded because of incomplete nationwide coverage of PALGA in some of the municipalities included in the Netherlands Cohort Study on Diet and Cancer during that period. Within this period, 83 subcohort members died or were diagnosed with cancer other than nonmelanoma skin cancer, leaving 3,263 subcohort members for analysis. From 1989 to 1994, 929 incident cases of histologically confirmed colorectal cancer were identified within the cohort, of which 819 could also be linked to a PALGA report of the lesion (figure 1). The PALGA reports were used to identify and locate tumor tissue from eligible colorectal cancer patients in Dutch pathology laboratories.
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APC mutation analysis
The majority of somatic mutations in the APC gene occur within the mutation cluster region (codons 12861520). Analysis of specific mutations in the mutation cluster region was performed on archival adenocarcinoma DNA by amplification of the mutation cluster region in four overlapping DNA fragments in a nested polymerase chain reaction (PCR), followed by direct sequencing of purified fragments as previously described (14). The detection limit was 5 percent mutated DNA, and duplicate experiments revealed good reproducibility (85 percent) (14
). From 72 of the 737 patients with a sufficient DNA yield, one or more fragments of the mutation cluster region could not be amplified; these patients were excluded from this study, leaving 665 patients (90 percent) available for subsequent analyses (figure 1).
hMLH1 immunohistochemistry
Formalin-fixed, paraffin-embedded tissues were sectioned at 4 µm and included tumor tissue with normal adjacent mucosa. Endogenous peroxidase activity was blocked with 3 percent hydrogen peroxide. Slides were submitted to microwave antigen retrieval in 1 mM ethylenediaminetetraacetic acid buffer (pH 8.0) and incubated with 10 percent normal horse serum for 10 minutes at room temperature. Sections were then incubated overnight at 4°C with mouse monoclonal antibodies against hMLH1 protein (clone G168-15; PharMingen, San Diego, California) at a 1:100 dilution. Antibody binding was detected by incubating the sections at room temperature with the peroxidase-labeled DAKO Envision System (DAKO, Carpinteris, California) and using diaminobenzidine as a chromogen. Sections were counterstained with hematoxylin.
Lesions were considered to lack hMLH1 protein expression when unequivocal absence of nuclear staining of the tumor epithelial cells was observed. Nuclear staining of normal epithelial and stromal cells or lymphocytes served as an internal positive control. Staining profiles were scored independently by at least two observers and, in the case of discordant results, discussed with a pathologist until consensus was reached. hMLH1 expression could be determined in 724 of the 737 patients for whom tumor DNA was available.
GSTM1 and GSTT1 polymorphism analyses
A seminested multiplex PCR was performed for simultaneous assessment of the presence or absence of GSTM1 and GSTT1 alleles. Flank PCR was performed to generate PCR products for GSTM1 and GSTT1 alleles and ß-globin using DNA derived from formalin-fixed paraffin-embedded tumor tissue. In a 1:100 dilution, the flank PCR product served as a template for a second round of PCR. In each PCR, one round of 35 cycles was performed. Series positive (ß-globin amplification) and negative (no DNA) controls were included in each PCR. The absence or presence and length of PCR products of GSTM1 and GSTT1 alleles were checked by electrophoresis on 2 percent agarose gels and visualized with ethidium bromide. Reproducibility was shown to be 91 percent (42/46).
Genotypes of GSTM1 or GSTT1 could not be determined in 79 patients for whom tumor DNA was available. These patients were excluded from this study.
Exposure assessment
In the questionnaire, tobacco smoking was addressed through questions on ages at first and last exposure to smoking, smoking frequency, smoking duration, and inhalation for cigarette smokers, cigar smokers, and pipe smokers. The dietary section of the 150-item semiquantitative food frequency questionnaire concentrated on habitual intake of foods and beverages, as well as lifestyle factors, during the year preceding the start of the study.
Questionnaire data were key-entered twice and processed for all incident cases in the cohort and for all subcohort members in a manner blinded with respect to case/subcohort status. Subjects whose dietary data were incomplete or inconsistent (49 cases, two of whom were also subcohort members, and 216 subcohort members) were excluded from the analyses. Criteria used for this selection were 60 or more questionnaire items left blank, consumption of 35 or more food items less than once per month, and/or one or more item blocks (groups of items, such as beverages) left blank. Eventually, 685 colorectal cancer patients (389 men, 296 women), of whom 12 were also subcohort members, and 3,048 subcohort members (1,475 men and 1,573 women) were included in the analyses (figure 1).
Statistical analysis
The data analyses were conducted separately for overall colorectal cancer, colorectal cancer with and without a truncating APC mutation, tumors with a specific C:GT:A or G:C
T:A point mutation resulting in a stop codon, and colorectal cancer with and without hMLH1 expression.
Using the Mann-Whitney U test or Student's t test, we analyzed differences in mean values for age at baseline (years), alcohol intake (g/day), and body mass index (weight (kg)/height (m)2) between all colorectal cancer patients and subcohort members, between patients with and without a truncating mutation in the mutation cluster region of the APC gene, and between patients with absent and present hMLH1 expression. The distributions of sex, family history of colorectal cancer (yes/no), and leisure-time physical activity (<30, 3060, 6090, >90 minutes/day) between all colorectal cancer patients, patients with a truncating APC mutation, or patients with absent hMLH1 expression and subcohort members were tested using the 2 test.
Incidence rate ratios (IRRs) for overall colorectal cancer according to smoking frequency (cigarettes/day), smoking duration (years), age at starting smoking (years), time since smoking cessation (years), and corresponding continuous variables, in comparison with lifelong nonsmoking, were estimated using Cox proportional hazards regression models. In addition, associations were estimated for specific tumor molecular endpoints.
Standard errors were estimated using the robust Huber-White sandwich estimator to account for additional variance introduced by sampling from the cohort (33). The proportional hazards assumption was tested using the scaled Schoenfeld residuals (34
). Tests for dose-response trends over the different categories of smoking variables were estimated by fitting the ordinal exposure variables as continuous terms and were evaluated using the Wald test. For estimation of the p value for trend over the categories of age at starting smoking and years since smoking cessation and the corresponding continuous analyses, nonsmokers were excluded.
Age (years) at baseline; sex; family history of colorectal cancer (yes/no); intake of vegetables (g/day), fruit (g/day), alcohol (g/day), coffee (cups/day), meat (g/day), fat (g/day), dietary fiber (g/day), calcium (mg/day), and energy (kJ/day); body mass index (kg/m2); and leisure-time physical activity (<30, 3060, 6090, >90 minutes/day) were considered as potential confounders. Variables that were found to statistically significantly (p < 0.05) contribute to the multivariate model of overall colorectal cancer (age at baseline, sex, body mass index, and family history of colorectal cancer) by means of the Wald test or that showed a 10 percent influence on the IRR for colorectal cancer were included as covariates in the multivariate analyses. Adjustment for body mass index gave rise to missing values in 100 subcohort members, 13 colon cancer cases, and six rectal cancer cases (figure 1).
In order to estimate interaction with the GSTM1 and GSTT1 genotypes, a case-only design was applied. Odds ratios for interaction between the genotype at risk (GSTM1 or GSTT1 null genotype) and smoking variables were obtained using logistic regression modeling. Adjustment for family history of colorectal cancer did not alter assessed odds ratios.
All p values reported are for a two-sided test; p values less than 0.05 were considered statistically significant.
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RESULTS |
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Tests for interactions between sex and any of the smoking characteristics used in this study did not produce statistically significant results (p = 0.16 and p = 0.47 for smoking frequency and duration, respectively). Therefore, results are presented for men and women combined. When the association between smoking frequency and colorectal cancer was examined for men and women separately, no association was observed in women, but IRRs for men were similar to the observed overall IRRs.
When we considered APC mutation status, introduction of a stop codon did not appear to be associated with any of the smoking variables (table 2). Moreover, specific point mutations in APC that would lead to the introduction of a stop codon and could be expected to be associated with cigarette smoking (i.e., G:CA:T transitions (65 patients) and G:C
T:A transversions (34 patients)) were also not associated with any of the smoking variables, with two exceptions: The categorical variable for age at starting smoking was inversely associated with G:C
A:T mutations (p for trend = 0.03), but no statistically significant association was observed for the continuous variable (data not shown). Smoking cessation was associated with a reduced risk of colorectal tumors harboring a G:C
T:A mutation (per 10 years of smoking cessation, IRR = 0.53, 95 percent CI: 0.28, 0.99). In contrast, tumors not harboring a truncating APC mutation appeared to be associated with former smoking but not with current smoking (for ex-smokers vs. never smokers, IRR = 1.49, 95 percent CI: 1.10, 2.00; for current smokers vs. never smokers, IRR = 0.85, 95 percent CI: 0.61, 1.18) or smoking frequency (per five-cigarette/day increment, IRR = 1.11, 95 percent CI: 1.05, 1.17).
Absent hMLH1 expression appears to occur almost exclusively in patients without a truncating APC mutation. Of 56 patients with absent hMLH1 expression, only five also harbored a truncating APC mutation. When estimating associations for smoking variables in the limited group of patients with a tumor lacking hMLH1 expression, positive associations, though not statistically significant, were found for smoking status (for ex-smokers vs. never smokers, IRR = 1.48, 95 percent CI: 0.72, 3.05; for current smokers vs. never smokers, IRR = 1.79, 95 percent CI: 0.90, 3.54), smoking frequency (per five-cigarette/day increment, IRR = 1.09, 95 percent CI: 0.98, 1.21), smoking duration (per 10-year increment, IRR = 1.17, 95 percent CI: 1.00, 1.37), and inhalation (for inhalation vs. never smoking, IRR = 1.94, 95 percent CI: 0.94, 3.99) (table 2). When smoking frequency and smoking duration were simultaneously included in the analysis, risk ratios were smaller, and smoking duration was found to be more influential than smoking frequency (per five-cigarette/day increment in frequency, IRR = 1.03, 95 percent CI: 0.91, 1.16; per 10-year increment in duration, IRR = 1.15, 95 percent CI: 0.97, 1.38).
Among patients with tumors that expressed hMLH1, a similar, statistically significant positive association with smoking frequency was observed (per five-cigarette/day increment, IRR = 1.07, 95 percent CI: 1.02, 1.22), but no association with smoking duration was observed (per 10-year increment, IRR = 1.00, 95 percent CI: 0.95, 1.06).
Table 3 presents odds ratios for interaction between the GSTM1 and GSTT1 null genotypes and all smoking variables for overall colorectal cancer, as determined in the case-only design. A borderline-significant (p = 0.06) interaction was observed between the GSTM1 null genotype and smoking frequency (for GSTM1 absence and an increment of five cigarettes/day, the odds ratio for interaction was 1.07 (95 percent CI: 1.00, 1.14)) but not with the GSTT1 null genotype and any of the smoking characteristics.
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DISCUSSION |
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In the questionnaire used in this study, questions retrospectively covered a period long enough for detection of possible associations, since the suggested induction time of 3045 years (3) was met in most cases. The relative importance of dose and duration of cigarette smoking remains somewhat in question, because these parameters are correlated. In this study, correlation between the two characteristics was found to be rather low (r = 0.22), and correcting smoking frequency for duration did not alter risk estimates much. For the association between cigarette smoking and overall colorectal cancer, frequency appeared to be more influential than duration. This is in accordance with previous studies that have also found that dose, or amount usually smoked per day, is an important predictor of risk (35
). It seems plausible that the absolute daily quantity of carcinogens from cigarettes delivered to the colorectal epithelium is important in producing mutations at a sufficiently high rate.
In accordance with a case-control study in which no association between cigarette smoking and truncating APC mutations was observed (23), we did not observe an association between cigarette smoking and truncating mutations in the APC gene. The associations observed for specific point mutations leading to truncation may be chance findings and require confirmation in another study.
Cigarette smoking appeared to be associated with tumors without a truncating APC mutation. Some tumors that do not harbor truncating APC mutations lack hMLH1 expression. We observed positive associations between smoking frequency and (especially) smoking duration and colorectal cancer without hMLH1 expression. This is in agreement with case-control studies that have found cigarette smoking to be associated with microsatellite instability in colon (20) and colorectal (21
, 22
) tumors but not with another report (23
). Cigarette smoking may directly affect the methylation of the hMLH1 promoter region, resulting in absent hMLH1 expression and hence deficient DNA repair (21
). On the other hand, cigarette smoke contains numerous carcinogenic compounds that may cause mutations. When DNA repair mechanisms fail (e.g., by lack of hMLH1 expression), tumors may become more susceptible to mutations that could eventually lead to carcinoma.
Polymorphisms in the genotype of the carcinogen metabolizing glutathione-S-transferases may alter colorectal cancer risk. One study found a borderline-significant twofold elevated risk associated with the GSTM1 null genotype and cigarette smoking in comparison with never smokers who had the GSTM1 present genotype for rectal cancer in men (28) but not for colon cancer (26
). No interaction was found for GSTM1 in colon adenomas (24
) or for GSTM1 and GSTT1 in colorectal cancer (25
, 27
).
A valid interpretation of the interaction parameter as estimated from a case-only study requires the assumption of independence between the gene polymorphism and exposure in the population (36, 37
). In a large group of controls from the database of the International Collaborative Study on Genetic Susceptibility to Environmental Carcinogens, it was shown that there was no association between smoking and the GSTM1 or GSTT1 genotype (38
). We did not observe statistically significant interactions between GSTM1 or GSTT1 and any of the smoking variables. A borderline-significant interaction between smoking frequency and GSTM1 genotype was observed. Although the odds ratio for interaction between the GSTM1 genotype and smoking frequency is difficult to interpret in the absence of information on the colorectal cancer risk conferred by the GSTM1 null genotype per se, the effect of GSTM1 genotype on cigarette smoking seems negligible. Because of the limited number of tumors displaying hMLH1 deficiency, no interaction of the GSTM1 and GSTT1 genotypes in this potentially interesting group could be estimated.
Although associations studied here were hypothesis-based, studying the different subsets of tumors resulted in multiple testing, which may have given rise to chance findings. The statistically significant results observed for overall colorectal cancer in this study are in accordance with results from other studies (3). The absence of hMLH1 is a rare occurrence in sporadic colorectal cancer; therefore, just 53 patients were available in our reasonably large cohort for studying associations between cigarette smoking and risk of colorectal tumors without hMLH1 expression. This limited number of patients may have given rise to spurious findings, but the observed associations for hMLH1-deficient tumors are also in keeping with previous reports on colon cancer (20
22
).
This study indicates that cigarette smoking may contribute to colorectal cancer. The association between frequency of cigarette smoking and colorectal cancer is most apparent in cancers that do not harbor a truncating mutation in the APC gene. Duration of smoking was associated with hMLH1-deficient tumors, though estimated risk ratios were moderate and their corresponding confidence intervals were wide. Finally, modification of the effect of cigarette smoking by the GSTM1 and GSTT1 null genotypes seems negligible.
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References |
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