1 Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Department of Epidemiology, Maastricht University, PO Box 616, 6200 MD, Maastricht, 2 Research Institute Growth and Development (GROW), Department of Pathology, Maastricht University, 3 NUTRIM, Department of Pathology, Maastricht University, and 4 TNO Nutrition and Food Research, PO Box 360, 3700 AJ Zeist, The Netherlands
5 To whom correspondence should be addressed Email: mp.weijenberg{at}epid.unimaas.nl
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Abstract |
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Abbreviations: CI, confidence intervals; CRC, colorectal cancer; MDA, malondialdehyde; M1G, pyrimidopurinonedeoxyguanosine adducts; MUFA, monounsaturated fat; NLCS, The Netherlands Cohort Study; RR, rate ratios; PUFA, polyunsaturated fat
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Introduction |
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The majority of colon and rectal tumours develop from small adenomatous polyps through a well-defined sequence of cytological and morphological changes (3), a process that is associated with the acquisition of somatic mutations (4,5). A genetic alteration that occurs in adenomas (10%) as well as carcinomas (40%) in colon and rectal cancer is the oncogenic activation of the K-ras gene by mutations. Activating mutations are mainly found in codons 12 and 13 (4,68). The most frequently observed types of point mutations are G > A transitions (8,9), and G > T and G > C transversions (10).
The link between fat intake and the pattern of mutations in human colon and rectal cancer is not clear. Only a few epidemiological studies have been conducted up to date on the association between the intake of fat and K-ras mutation status (1114) and results are inconsistent. Experimental studies suggest that peroxidation of -6 polyunsaturated fatty acids (PUFAs) could lead to the accumulation of by-products like malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). These compounds could react with DNA to form adducts, mainly the pyrimidopurinone adduct of deoxyguanosine (M1G) (1517). The presence of the M1G adducts resulted predominantly in G > A and G > T, with very few G > C transversions in bacteria (16,18,19).
Consequently, exposures to specific dietary fat and fatty acids could contribute to the heterogeneity of acquired genetic alterations in the K-ras oncogene observed in colon and rectal tumours. Associations between dietary intakes of fat and specific fatty acids and the risk of specific point mutations in the K-ras oncogene in patients with colon and rectal cancer were studied within the framework of The Netherlands Cohort Study on diet and cancer (NLCS).
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Materials and methods |
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The first 2.3 years of follow up were excluded due to incomplete coverage of PALGA alone in some of the municipalities included in the NLCS. Within this period, 83 subcohort members were either deceased or diagnosed with cancer other than non-melanoma skin cancer, leaving 3263 men and women for analysis. From 1989 until 1994, 929 incident cases with histologically confirmed CRC were observed of whom 819 could also be linked to a PALGA report of the lesion. The PALGA database was used to identify and locate tumour tissue in Dutch pathology laboratories. CRC was classified according to site as follows: colon, i.e. cecum through sigmoid colon (ICD-O-1 codes: 153.0, 153.1, 153.2, 153.3, 153.4, 153.5, 153.6, 153.7, 153.8, 153.9), rectosigmoid (ICD-O-1 code 154.0) and rectum (ICD-O-1 code 154.1). Information about age at baseline, sex and family history of CRC (at baseline) was retrieved from the NLCS database.
Tissue samples
This study is based on data of gene mutation analysis from CRC patients, described in detail elsewhere (8). Briefly, tumour material of all CRC patients was collected after approval by the Medical Ethics Committees of Maastricht University, the NCR and PALGA. Subsequently, all pathology laboratories in The Netherlands agreed to make relevant tissue samples available upon request from PALGA. Tissue samples of the 819 cases were distributed among 54 pathology laboratories throughout The Netherlands. Tumour tissue specimen collection started in August 1999 and was completed in December 2001. The loss to follow-up of tissue samples of cases amounted to 5%. Tissue samples from nine patients registered in one pathology laboratory could not be retrieved due to administrative inconsistencies, leaving 810 tissue samples for collection. For 34 cases, paraffin-embedded material was not available in the archives of pathology laboratories, leaving 776 cases for the determination of the K-ras mutation status. For 39 cases (5%), the K-ras mutation status could not be determined, i.e. for 20 cases only normal colonic mucosa was available, 10 cases were revised with a benign adenoma instead of an adenocarcinoma, for six cases the yield of DNA was not sufficient enough to determine K-ras mutation status and for three cases the available tissue did not include malignant CRC tissue. Finally, tumour material from 737 incident colorectal adenocarcinoma cases was available of whom 476 were colon cancer cases, 85 were rectosigmoid cancer cases and 176 were rectal cancer cases. Statistical analyses were performed separately for colon and rectal cancer as differences in the aetiology of colon and rectal cancer have been reported (1). Since the rectosigmoid can be considered as a clinically applied term rather than an anatomically defined transitional zone between the colon and rectum, patients with a rectosigmoid tumour were excluded from data analyses. Moreover, the number of patients with a rectosigmoid tumour was too small for adequate stratified analyses (8).
Detection of K-ras mutations
Mutation analysis of the exon 1 fragment of the K-ras oncogene, spanning codons 829, was performed on archival colorectal adenocarcinoma specimens of all 737 CRC patients using macrodissection, nested polymerase chain reaction (PCR) and direct sequencing of purified fragments, which has been described in detail elsewhere (8). The method of mutation detection was validated by the confirmation of reported K-ras status in CRC cell lines and a good correlation between fresh-frozen and routinely fixed, paraffin-embedded tissue. The detection limit was 5% mutated DNA. Duplo analyses revealed a good reproducibility (88%) (8). Evaluation of mutation analysis and data entry was independently performed by two observers (G.R. and M.L.).
The food frequency questionnaire
The dietary section of the questionnaire was a 150-item semi-quantitative food frequency questionnaire, which concentrated on habitual consumption of food and beverages during the year preceding the start of the study. Daily mean nutrient intakes were calculated using the computerized Dutch food composition table (26), by cumulating the multiplied frequencies and portion sizes of all food items with their tabulated nutrient contents. The questionnaire was validated against a 9-day diet record (27). Crude and energy-gender-adjusted (in parentheses) correlation coefficients were 0.72 (0.52) for total fat, 0.73 (0.58) for saturated fat and 0.73 (0.75) for PUFA (27). For energy intake the correlation coefficient was 0.74. On average, the questionnaire covered 91% of the record intake explaining part of the underestimation of energy intake from the questionnaire data (as presented in Table I). 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. This was done in order to minimize observer bias in coding and interpretation of the data.
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Intake of specific fatty acids was based on a food composition database with specific fatty acids derived from the TRANSFAIR study (28). For this database, the hundred foods that contributed most to fat intake in the Dutch dietary pattern were sampled and analysed as methyl esters of the fatty acids present in the foods. In the database, total fat includes triglycerides and other lipids such as phospholipids and sterols. The percentage of triglycerides in total fat is assumed to be on average 93%, but varies across food sources. Daily intakes of total fat (g/day), saturated fat (g/day), monounsaturated fat (MUFA) (g/day), PUFA (g/day), and linolenic acid (g/day) and linoleic acid (g/day) as main constituents of PUFA were used as exposure variables. Linolenic and linoleic acid were used as the most abundant sources of -3 PUFAs and
-6 PUFAs. For data-analyses, quartiles of the intake of fat and fatty acids were computed based on the distribution of subcohort members. Daily intake of dietary fibre (g/day), alcohol (g/day), fruit (g/day), vegetables (g/day) and total energy (kcal/day) and age at baseline (years), sex (men/women), Quetelet Index (QI; kg/m2), physical activity (<30 min/day, 3060 min/day, 6090 min/day, >90 min/day), family history of CRC (yes/no) and smoking status (never/ex/current) were regarded as potential confounders.
Statistical analysis
The overall frequency of K-ras mutations as well as the type of mutation was computed for all colon and rectal cancer cases as described elsewhere (8). Fat intake was adjusted for energy by the residual method (29). Mean values of the continuous variables age at baseline (years), intake of total fat, saturated fat, MUFA, PUFA, linolenic acid, linoleic acid, dietary fibre, alcohol, fruit, vegetables and total energy and QI were evaluated for subcohort members and colon and rectal cancer cases with wild-type and mutated K-ras gene. Distributions in the categorical variables sex, family history of CRC, smoking status and physical activity were evaluated for subcohort members and colon and rectal cancer patients with wild-type and mutated K-ras gene. Differences in mean values of the continuous variables and the distributions in categorical variables between patients with wild-type and mutated K-ras tumours were tested with the Student's t-test and 2-test, respectively, using the statistical software package SPSS (version 9).
Incidence rate ratios (RR) and corresponding 95% confidence intervals (CI) for colon and rectal cancer cases with wild-type or mutated K-ras gene tumours were estimated according to intakes of quartiles (with the lower quartile of intake regarded as the reference group) and of 1 standard deviation (SD) of increase of intake of total fat, saturated fat, MUFA, PUFA, linolenic acid and linoleic acid using Cox proportional hazards models with the STATA statistical software package (intercooled STATA, version 7). The total person-years at risk, estimated from the subcohort, were used in the analyses (27). Standard errors were estimated using the robust HubertWhite sandwich estimator to account for additional variance introduced by sampling from the cohort. This method is equivalent to the variancecovariance estimator as presented by Barlow (30). The proportional hazards assumption was tested using the scaled Schoenfeld residuals (31). Using the backwards stepwise procedure, all confounders were tested in the overall colon and rectal cancer models separately. Interactions between fat and specific fatty acid intake and sex were tested for colon and rectal cancer separately and never found to be statistically significant. Therefore, results for men and women are presented together. Finally, age at baseline, sex, family history of CRC, smoking status, QI and the intake of energy were confounders for either one or both of the models and were therefore included as covariates for all models to be tested. Since 100 subcohort members had missing values for QI, results in the tables will be presented for 2948 subcohort members. For each analysis, trends were evaluated with the Wald test by fitting ordinal exposure variables (quartiles of intake) as continuous terms.
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Results |
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Table I shows various types of fat intake and other baseline characteristics of the study population. Colon and rectal cancer cases were more often men, were older, more frequently reported a family history of colorectal cancer and had a higher daily alcohol intake as compared with the subcohort. Colon cancer cases with a mutated K-ras tumour were significantly older, had higher daily intakes of PUFA, linoleic acid, fibres and vegetables than colon cancer cases with a wild-type K-ras tumour. There were no statistically significant differences between colon cancer cases with a wild-type K-ras tumour and a mutated K-ras tumour in dietary intakes of total fat, saturated fat, MUFA, linolenic acid and other factors presented in Table I. Rectal cancer cases with a mutated K-ras tumour were less frequently men as compared with rectal cancer cases with a wild-type K-ras tumour. No statistically significant differences between rectal cancer cases with mutated K-ras tumours and wild-type K-ras tumours were observed for total fat, specific fatty acids and other (dietary) factors.
Associations between the intake of total fat, saturated fat, MUFA, PUFA, linolenic acid and linoleic acid with the risk of colon or rectal cancer are presented in Table II. Incidence RR and 95% CI for colon and rectal cancer are adjusted for age and sex and for age, sex, smoking, QI, energy intake and family history of CRC. The agesex adjusted RR and the multivariate RR were similar. Frequent consumption of total fat, saturated fat, MUFA, PUFA, linolenic acid and linoleic acid were all not associated with the risk of colon or rectal cancer (Table II).
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Discussion |
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This cohort study on fat and specific fatty acids intake in relation to specific K-ras mutations in colon and rectal cancer is, to our knowledge, the only prospective study performed to date. One large case-control study with colon cancer patients (11), two case-control studies of limited size among colon (13), and colon and rectal cancer patients (12) and one cross-sectional casecase study with colorectal adenoma patients (14) have been conducted previously. The prospective design of the current study and high completeness of follow-up of cancer incidence and subcohort, make information and selection bias unlikely. In addition, as a result of the exclusion of the first 2.3 years of follow-up, the chance of information bias due to potential pre-clinical colorectal cancer is minimal. Finally, tumour tissue was available for 84% of the eligible cases (776 out of 929) and there were no significant differences in fat intake levels nor in clinicopathological characteristics of the tumour (Dukes' stage and differentiation grade) of these patients compared with the CRC patients with a known K-ras mutation status of their tumour (data not shown). Therefore, selection bias due to loss to follow-up is unlikely.
In general, the validation of the fat intake variables was satisfactory (see Materials and methods section and ref. 27). Although total fat intake and energy intake may have been underestimated as compared with the 9-day record method in the validation study (27) and, in addition, there is inevitably a certain degree of misclassification of fat intake variables, it is not expected that this is differential with respect to the K-ras mutation status of the tumour. Therefore, the associations observed in this study are probably attenuated. In other epidemiological studies similar inaccuracies occur in the measurement of fat intake since similar methods of intake assessment were employed (11,12,14).
One of the hypotheses for the association between fat intake and colorectal cancer is that meat consumption instead of fat intake determines the increased risk. We also investigated meat consumption as a potential risk factor for K-ras mutations in colon and rectal cancer, and total fresh meat was not associated with risk. Therefore, we did not additionally adjust for this potential confounder in these analyses.
In the current study, a high intake of total and saturated fats was not associated with overall colon or rectal cancer risk and with K-ras mutation status and these findings are supported by others (11,13,14). Bautista et al. (12) observed an inverse association between a high intake of total fat and colorectal tumours with a K-ras wild-type gene (odds ratio for highest versus lowest tertile of intake 0.47, 95% CI 0.220.99). However, the results were based on only 108 colorectal cancer cases in a population-based case-control setting, and therefore estimates of associations must be interpreted with caution.
Animal studies indicated an inverse association of high MUFA intake, derived mainly from oleic acid with colorectal cancer risk (32,33). However, in epidemiological studies, no clear associations were observed with colorectal cancer risk (3436). Regarding the K-ras mutation status, no associations were observed for high intake of MUFA and the risk of colon and rectal cancer in the current study. This is in line with Slattery's observation (11). However, Bautista et al. (12) observed that a high intake of MUFA was inversely associated with the risk of having a wild-type K-ras gene. This study was conducted in Spain where the main source of oleic acid is olive oil. This is not the case for the current cohort and could be an explanation for the different findings. The association was not reported in the other studies (13,14).
To our best knowledge, this is the first study reporting on specific PUFAs in relation to K-ras mutation status. Overall PUFA intake was studied by Slattery et al. (11) and Bautista et al. (12) and no associations were observed with K-ras mutation status.
In the current study, no clear associations were observed between the high intake of linolenic acid as the main source of -3 PUFAs, and the risk of colon or rectal cancer with or without specific point mutations in the K-ras gene. Epidemiological, clinical and experimental data indicate a protective effect of fish oil-derived
-3 PUFAs on overall colon cancer (35,3744). Collett et al. (37) demonstrated that the major
-3 PUFA constituent of fish oil, docosahexaenoic acid reduces the Ras protein localization to the plasma membrane without affecting post-translational lipidation and lowers the GTP-binding of the Ras protein in mouse colonocytes treated with azoxymethane (colon carcinogen). These findings were corroborated by in vivo studies (45). Another hypothesis is that the tumour-promoting activity of
-6 PUFA was abrogated by competitive inhibition of
-3 PUFA from the metabolism of arachidonic acid and therefore reducing MDA levels and subsequently M1G > T and M1G > C transversions (38). Summarized,
-3 PUFAs may exert their effect through inhibition of the Ras protein activity and not by generating functional aberrations in the exon 1 fragment of the K-ras oncogene. This could explain the lack of association between linolenic acid and K-ras mutation status of colon or rectal tumours in this study.
In the current study, no significant association was observed for linoleic acid, as the main source of -6 PUFAs intake, and the overall risk of colon or rectal cancer. This is in line with most epidemiological evidence up to date (46). However, a positive effect was found between high intake of linoleic acid and colon tumours harbouring K-ras gene mutations. Subgroup analyses of specific point mutations in the K-ras gene revealed that colon tumours harbouring G > A transitions or G > T or G > C transversions are positively associated with high linoleic acid intake. These associations are in line with the biological evidence (16,18) and are possibly the result of an increased formation of G-adducts resulting in G > A transitions or G > T or G > C transversions. Increased intake of
-6 PUFAs enhance tumorigenesis in experimental animals and in vitro systems by several mechanisms (47). The conjugated double bonds in PUFAs are highly sensitive to lipid peroxidation, and may generate fatty acid hydroperoxides. These hydroperoxides will be reduced by glutathione peroxidase to non-reactive fatty acid alcohols or may react with metal ions to yield alkoxyl radicals. The fatty acid alcohols do not lead to DNA damage and will not be discussed further. However, alkoxyl radicals are reduced to aldehydes, with 2,3-epoxy-4-hydroxynonanal (4-HNE) and MDA as the most prominent forms. The bifunctional alkylating agent 4-HNE can react with DNA to yield etheno and other base adducts which are thought to promote the carcinogenic process. Etheno-dG induces mainly transitions to A and transversions to T in bacteria (16,33,48,49) and etheno-dC induces transitions to T in the kidneys of monkeys (50). MDA is a major genotoxic carbonyl compound, which is also a by-product of the arachidonic acid metabolism in the synthesis of prostaglandins. MDA is mutagenic in bacterial and mammalian cells, and carcinogenic in rats (18). MDA reacts with DNA to form adducts, predominantly with deoxyguanosine to generate M1G (15). The most common mutations in progeny molecules are G > T transversions and G > A transitions, with a minor contribution by G > C transversions (16,18,19). In the current study, the associations with G > A transitions, G > T and G > C transversions in the K-ras oncogene were only observed for colon cancer patients and not for rectal cancer patients. The reason for this apparent difference in the aetiology remains unclear. In addition, the observed significant association found for colon cancer could be a result of a type I error due to the numerous associations analysed in this study. Although the studied associations were mainly hypotheses-driven, caution is warranted in interpreting these results and further investigations are needed.
Our results suggest that PUFA, and in particular linoleic acid, is an important dietary risk factor for colon tumours with K-ras mutations possibly by generating G > A transitions or G > T or G > C transversions in the exon 1 fragment of the K-ras oncogene. This implies that, for some dietary exposures like polyunsaturated fat intake, it is meaningful to account for somatic mutations in the K-ras oncogene in the aetiology of colon and rectal cancer.
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Acknowledgments |
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References |
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