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Folate and Vitamin B6 Intake and Risk of Colon Cancer in Relation to p53 Expression

2008; Elsevier BV; Volume: 135; Issue: 3 Linguagem: Inglês

10.1053/j.gastro.2008.06.033

1528-0012

Eva Schernhammer, Shuji Ogino, Charles S. Fuchs,

Nutritional Studies and Diet

Background & Aims: Considerable evidence suggests that a low-folate diet increases the risk of colorectal cancer, although the results of a recent randomized trial indicate that folate supplementation may not reduce the risk of adenoma recurrence. In laboratory models, folate deficiency appears to induce p53 mutation. Methods: We immunohistochemically assayed p53 expression in paraffin-fixed colon cancer specimens in a large prospective cohort of women with 22 years of follow-up to examine the relationship of folate intake and intake of other one-carbon nutrients to risks by tumor p53 expression. Results: A total of 399 incident colon cancers accessible for p53 expression were available. The effect of folate differed significantly according to p53 expression (Pheterogeneity = .01). Compared with women reporting folate intake <200 μg/day, the multivariate relative risks (RRs) for p53-overexpressing (mutated) cancers were 0.54 (95% confidence interval [CI], 0.36–0.81) for women who consumed 200–299 μg/day, 0.42 (95% CI, 0.24–0.76) for women who consumed 300–399 μg/day, and 0.54 (95% CI, 0.35–0.83) for women who consumed ≥400 μg/day. In contrast, total folate intake had no influence on wild-type tumors (RR, 1.05; 95% CI, 0.73–1.51; comparing ≥400 with <200 μg/day). Similarly, high vitamin B6 intake conferred a protective effect on p53-overexpressing cancers (top versus bottom quintile: RR, 0.57; 95% CI, 0.35–0.94; Pheterogeneity = .01) but had no effect on p53 wild-type tumors. Conclusions: We found that low folate and vitamin B6 intake was associated with an increased risk of p53-overexpressing colon cancers but not wild-type tumors. Background & Aims: Considerable evidence suggests that a low-folate diet increases the risk of colorectal cancer, although the results of a recent randomized trial indicate that folate supplementation may not reduce the risk of adenoma recurrence. In laboratory models, folate deficiency appears to induce p53 mutation. Methods: We immunohistochemically assayed p53 expression in paraffin-fixed colon cancer specimens in a large prospective cohort of women with 22 years of follow-up to examine the relationship of folate intake and intake of other one-carbon nutrients to risks by tumor p53 expression. Results: A total of 399 incident colon cancers accessible for p53 expression were available. The effect of folate differed significantly according to p53 expression (Pheterogeneity = .01). Compared with women reporting folate intake <200 μg/day, the multivariate relative risks (RRs) for p53-overexpressing (mutated) cancers were 0.54 (95% confidence interval [CI], 0.36–0.81) for women who consumed 200–299 μg/day, 0.42 (95% CI, 0.24–0.76) for women who consumed 300–399 μg/day, and 0.54 (95% CI, 0.35–0.83) for women who consumed ≥400 μg/day. In contrast, total folate intake had no influence on wild-type tumors (RR, 1.05; 95% CI, 0.73–1.51; comparing ≥400 with <200 μg/day). Similarly, high vitamin B6 intake conferred a protective effect on p53-overexpressing cancers (top versus bottom quintile: RR, 0.57; 95% CI, 0.35–0.94; Pheterogeneity = .01) but had no effect on p53 wild-type tumors. Conclusions: We found that low folate and vitamin B6 intake was associated with an increased risk of p53-overexpressing colon cancers but not wild-type tumors. See Pendergrass CJ et al on page 1011 in CGH. See Pendergrass CJ et al on page 1011 in CGH. Folic acid and related vitamins B2, B6, and B12 are essential for DNA methylation and the production of purine and pyrimidine nucleotides required for DNA synthesis. Considerable epidemiologic evidence suggests that a low-folate diet is associated with an increased risk of colorectal cancer.1Kim Y.I. Folate, colorectal carcinogenesis, and DNA methylation: lessons from animal studies.Environ Mol Mutagen. 2004; 44: 10-25Crossref PubMed Scopus (160) Google Scholar, 2Giovannucci E. Epidemiologic studies of folate and colorectal neoplasia: a review.J Nutr. 2002; 132: 2350S-2355SPubMed Google Scholar, 3Harnack L. Jacobs Jr, D.R. Nicodemus K. et al.Relationship of folate, vitamin B-6, vitamin B-12, and methionine intake to incidence of colorectal cancers.Nutr Cancer. 2002; 43: 152-158Crossref PubMed Scopus (122) Google Scholar Animal data support an antineoplastic effect of folate; however, in some animal studies, folate deficiency protects against, and supplementation increases, experimental carcinogenesis.1Kim Y.I. Folate, colorectal carcinogenesis, and DNA methylation: lessons from animal studies.Environ Mol Mutagen. 2004; 44: 10-25Crossref PubMed Scopus (160) Google Scholar In addition, a distinction between natural folate and synthetic folic acid must be made given the higher bioavailability of folic acid and a growing body of evidence suggesting that too much of the synthetic folic acid might be harmful.4Ulrich C.M. Folate and cancer prevention: a closer look at a complex picture.Am J Clin Nutr. 2007; 86: 271-273PubMed Scopus (112) Google Scholar Nevertheless, on the whole, the biological and epidemiologic evidence is consistent with the hypothesis that folate deficiency increases the risk of colorectal neoplasia in humans. The mechanism by which folate deficiency influences colon cancer risk remains less well understood. Folate deficiency leads to mutations and chromosomal damage, effects that are also central to the efficacy of antifolate chemotherapeutic agents (such as methotrexate). Specifically, animal studies suggest that folate deficiency can induce DNA strand breaks in a highly conserved region of the p53 tumor suppressor gene.5Kim Y.I. Shirwadkar S. Choi S.W. et al.Effects of dietary folate on DNA strand breaks within mutation-prone exons of the p53 gene in rat colon.Gastroenterology. 2000; 119: 151-161Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 6Crott J.W. Choi S.W. Ordovas J.M. et al.Effects of dietary folate and aging on gene expression in the colonic mucosa of rats: implications for carcinogenesis.Carcinogenesis. 2004; 25: 69-76Crossref PubMed Scopus (46) Google Scholar, 7Sohn K.J. Stempak J.M. Reid S. et al.The effect of dietary folate on genomic and p53-specific DNA methylation in rat colon.Carcinogenesis. 2003; 24: 81-90Crossref PubMed Scopus (90) Google Scholar In human colon cell lines and lymphocytes, folate depletion increased p53 expression and p53 strand breaks.8Crott J.W. Liu Z. Keyes M.K. et al.Moderate folate depletion modulates the expression of selected genes involved in cell cycle, intracellular signaling and folate uptake in human colonic epithelial cell lines.J Nutr Biochem. 2008; 19: 328-335Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 9Crott J.W. Liu Z. Choi S.W. et al.Folate depletion in human lymphocytes up-regulates p53 expression despite marked induction of strand breaks in exons 5–8 of the gene.Mutat Res. 2007; 626: 171-179Crossref PubMed Scopus (23) Google Scholar Further, folate depletion–induced hypomethylation of colon cells specifically in the region of the p53 gene was fully reversed by folic acid supplementation,10Wasson G.R. McGlynn A.P. McNulty H. et al.Global DNA and p53 region-specific hypomethylation in human colonic cells is induced by folate depletion and reversed by folate supplementation.J Nutr. 2006; 136: 2748-2753PubMed Scopus (90) Google Scholar suggesting that p53 mutations may play an important role in the effect of folate on colon cancer risk. The reported frequency of p53 mutations in colorectal cancer varies; whereas early reports suggested that 75% of colorectal cancers harbored p53 mutations, more recent estimates suggest that 40%–50% of tumors possess alterations in p53.11Vogelstein B. Fearon E.R. Hamilton S.R. et al.Genetic alterations during colorectal-tumor development.N Engl J Med. 1988; 319: 525-532Crossref PubMed Scopus (6144) Google Scholar, 12Baker S.J. Preisinger A.C. Jessup J.M. et al.p53 gene mutations occur in combination with 17p allelic deletions as late events in colorectal tumorigenesis.Cancer Res. 1990; 50: 7717-7722PubMed Google Scholar, 13Hollstein M. Rice K. Greenblatt M.S. et al.Database of p53 gene somatic mutations in human tumors and cell lines.Nucleic Acids Res. 1994; 22: 3551-3555PubMed Google Scholar We assessed whether the influence of folate on colon cancer risk differed according to p53 expression. For this purpose, we used tumor specimens from a prospective cohort study that previously showed that folate intake was inversely associated with the risk of colon cancer.14Giovannucci E. Stampfer M.J. Colditz G.A. et al.Multivitamin use, folate, and colon cancer in women in the Nurses' Health Study.Ann Intern Med. 1998; 129: 517-524Crossref PubMed Scopus (668) Google Scholar The availability of detailed and updated information on folate intake and tumor specimens within this cohort permitted a more comprehensive examination of the effect of folate intake on p53 expression status in colon cancers. Specificity in the association between folate and colon cancer to particular tumor markers would further enhance the case for causality and would provide important insights into the carcinogenic mechanisms of folate deficiency. The Nurses' Health Study was established in 1976 when 121,701 US female registered nurses aged 30–55 years completed a mailed questionnaire.15Chen J. Giovannucci E. Hankinson S.E. et al.A prospective study of methylenetetrahydrofolate reductase and methionine synthase gene polymorphisms, and risk of colorectal adenoma.Carcinogenesis. 1998; 19: 2129-2132Crossref PubMed Scopus (153) Google Scholar Follow-up within the cohort currently exceeds 92%. We mailed biennial questionnaires to update information and identify newly diagnosed cases of cancer. In 1980, the Nurses' Health Study questionnaire was expanded to include a validated assessment of diet, and updated dietary assessments have been conducted every 4 years.16Willett W.C. Sampson L. Stampfer M.J. et al.Reproducibility and validity of a semiquantitative food frequency questionnaire.Am J Epidemiol. 1985; 122: 51-65Crossref PubMed Scopus (3668) Google Scholar The institutional review boards at Brigham and Women's Hospital and the Harvard School of Public Health approved this study. Dietary intake of various nutrients, including folate, vitamin B6, vitamin B12, and methionine, was assessed by self-administered semiquantitative food frequency questionnaires, which were completed in 1980, 1984, 1986, 1990, 1994, and 1998. Nutrient intakes were calculated by multiplying the reported frequency of consumption of each specified food item by the nutrient content of the specified portion size and then summing these products for all food items. Information on multivitamin use and the use of other supplements was also collected, including details on which brand name and type. An extensive database of supplement formulation was then used to calculate specific nutrient contributions from these supplemental sources. These nutrient contributions were subsequently added to the specific nutrient intake from foods to calculate a daily intake for each woman. This method of dietary assessment has been extensively validated and its reliability evaluated.16Willett W.C. Sampson L. Stampfer M.J. et al.Reproducibility and validity of a semiquantitative food frequency questionnaire.Am J Epidemiol. 1985; 122: 51-65Crossref PubMed Scopus (3668) Google Scholar The correlation coefficient was 0.55 for the correlation between total folate intake calculated from the 1980 questionnaire and erythrocyte folate concentrations measured in 1987 in this cohort.17Giovannucci E. Stampfer M.J. Colditz G.A. et al.Folate, methionine, and alcohol intake and risk of colorectal adenoma.J Natl Cancer Inst. 1993; 85: 875-884Crossref PubMed Scopus (638) Google Scholar Moreover, vitamin B6 intake as assessed by 1980, 1984, and 1986 semiquantitative food frequency questionnaires has been shown to correlate with 1-week diet records, with correlations ranging from 0.54 to 0.58.16Willett W.C. Sampson L. Stampfer M.J. et al.Reproducibility and validity of a semiquantitative food frequency questionnaire.Am J Epidemiol. 1985; 122: 51-65Crossref PubMed Scopus (3668) Google Scholar, 18Willett W.C. Sampson L. Browne M.L. et al.The use of a self-administered questionnaire to assess diet four years in the past.Am J Epidemiol. 1988; 127: 188-199PubMed Google Scholar Alcohol consumption was the sum of the values for 3 types of beverages: beer, wine, and spirits. We assumed an ethanol content of 13.1 g for a 12-oz (38-dL) can or bottle of beer, 11.0 g for a 4-oz (12-dL) glass of wine, and 14.0 g for a standard portion of spirits. In validation studies, the correlation coefficient for the correlation between alcohol consumption derived from the 1980 semiquantitative food frequency questionnaire and the average of four 1-week diet records was 0.90.19Willett W.C. Stampfer M.J. Colditz G.A. et al.Moderate alcohol consumption and the risk of breast cancer.N Engl J Med. 1987; 316: 1174-1180Crossref PubMed Scopus (414) Google Scholar Other risk factors for colon cancer such as physical exercise and body mass index have generally been assessed biennially on the main questionnaires. We included colon cancers reported on the biennial questionnaires between the return of the 1980 questionnaire and June 1, 2002. With permission from study participants, colon cancer was confirmed through physicians' review of the nurses' medical records. If permission was denied, we attempted to confirm the self-reported cancer with an additional letter or telephone call. We also searched the National Death Index to identify deaths among the nonrespondents to each 2-year questionnaire. The computerized National Death Index is a highly sensitive method for identifying deaths in this cohort.20Stampfer M.J. Willett W.C. Speizer F.E. et al.Test of the National Death Index.Am J Epidemiol. 1984; 119: 837-839PubMed Google Scholar For all deaths attributable to colon cancer, we requested permission from family members (subject to state regulation) to review the medical records. Colon cancer was considered the cause of death if the medical records or autopsy reports confirmed fatal colon cancer or if colon cancer was listed as the underlying cause of death without another more plausible cause. Beginning in 2001, we began retrieving, from treating hospital pathology departments, representative pathologic specimens from participants whom we confirmed to have developed colon cancer. We successfully obtained specimens for 58% of cases (n = 527) over 22 years of follow-up in the Nurses' Health Study. To estimate p53 expression, we examined specimens for abnormalities of p53 protein accumulation by immunohistochemistry. This technique takes advantage of the phenomenon that mutant p53 proteins have a prolonged half-life in the nucleus due to a decreased rate of degradation.21Melhem M.F. Law J.C. el-Ashmawy L. et al.Assessment of sensitivity and specificity of immunohistochemical staining of p53 in lung and head and neck cancers.Am J Pathol. 1995; 146: 1170-1177PubMed Google Scholar, 22Nishio M. Koshikawa T. Kuroishi T. et al.Prognostic significance of abnormal p53 accumulation in primary, resected non-small-cell lung cancers.J Clin Oncol. 1996; 14: 497-502Crossref PubMed Scopus (147) Google Scholar Therefore, the increased nuclear staining is correlated with point mutations, which involve more than 80% of p53 alterations.23Greenblatt M.S. Bennett W.P. Hollstein M. et al.Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis.Cancer Res. 1994; 54: 4855-4878PubMed Google Scholar In contrast, immunohistochemistry will not detect frameshifts or mutations generating stop codons, which may involve only 8% of the p53 defects in human colorectal cancers.23Greenblatt M.S. Bennett W.P. Hollstein M. et al.Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis.Cancer Res. 1994; 54: 4855-4878PubMed Google Scholar Although the concordance between p53 overexpression and mutations is not perfect, the immunohistochemical assay is a rapid and cost-effective evaluation. In comparative studies, p53 overexpression is a good approximation of the real mutation rate, with approximately 90% concordance between immunostaining and gene analysis22Nishio M. Koshikawa T. Kuroishi T. et al.Prognostic significance of abnormal p53 accumulation in primary, resected non-small-cell lung cancers.J Clin Oncol. 1996; 14: 497-502Crossref PubMed Scopus (147) Google Scholar and 91% specificity.21Melhem M.F. Law J.C. el-Ashmawy L. et al.Assessment of sensitivity and specificity of immunohistochemical staining of p53 in lung and head and neck cancers.Am J Pathol. 1995; 146: 1170-1177PubMed Google Scholar We limited the current analysis to those participants for whom we were able to obtain sufficient amounts of tumor tissue paraffin blocks (n = 399; 76% of those for whom we successfully obtained a specimen). A random sample of 58 cancers was reread by the same pathologist 2 years apart, and the concordance was 0.95 (κ = 0.90; P < .001). Further, a random sample of 118 tumors was reexamined by a second observer (K. Nosho) who was unaware of other data. The concordance between the 2 observers was 0.87 (κ = 0.75; P < .0001), indicating substantial agreement. Baseline characteristics among 908 participants with colon cancer who we did and did not analyze for p53 expression were largely similar (all were white; mean age, 50.3 vs 50.8 years; former or current smoker, 58.7% vs 58.2%; mean body mass index, 25.1 vs 24.9 kg/m2; mean metabolic equivalents score per week, 12.0 vs 13.2; current multivitamin use, 31.3% vs 29.3%; folate intake, 352 vs 363 μg/day; calcium intake, 718 vs 707 mg/day; alcohol intake, 6.7 vs 7.0 g/day; P > .35 for all comparisons). Tissue microarray blocks were constructed for p53 analysis as previously described24Ogino S. Brahmandam M. Kawasaki T. et al.Combined analysis of COX-2 and p53 expressions reveals synergistic inverse correlations with microsatellite instability and CpG island methylator phenotype in colorectal cancer.Neoplasia. 2006; 8: 458-464Abstract Full Text PDF PubMed Scopus (74) Google Scholar using an automated arrayer (Beecher Instruments, Sun Prairie, WI). We examined 2–4 tumor tissue cores for p53 immunohistochemistry. A previous validation study has shown that examining 2 tissue microarray cores can yield comparable results to examining whole tissue sections in more than 95% of cases.25Camp R.L. Charette L.A. Rimm D.L. Validation of tissue microarray technology in breast carcinoma.Lab Invest. 2000; 80: 1943-1949Crossref PubMed Scopus (671) Google Scholar We have previously described in detail our method for p53 staining (immunohistochemistry).26Ogino S. Kawasaki T. Kirkner G.J. et al.Loss of nuclear p27 (CDKN1B/KIP1) in colorectal cancer is correlated with microsatellite instability and CIMP.Mod Pathol. 2007; 20: 15-22Crossref PubMed Scopus (52) Google Scholar We visually estimated the fraction of tumor cells with unequivocal strong nuclear staining for p53 by examining at least 2 tissue cores in tissue microarrays, or the whole tissue section in each case for which there was not enough tissue for tissue microarrays or results were equivocal in tissue microarrays. The presence of p53 overexpression (p53 positivity) was defined as 50% or more of tumor cells with unequivocal moderate/strong nuclear staining, as recommended for improved specificity.27Hall P.A. McCluggage W.G. Assessing p53 in clinical contexts: unlearned lessons and new perspectives.J Pathol. 2006; 208: 1-6Crossref PubMed Scopus (60) Google Scholar The absence of p53 expression (p53 negativity) was defined as either absent/weak staining or <50% of tumor cells with moderate/strong staining. Appropriate positive and negative controls were included in each run of immunohistochemistry. All immunohistochemically stained slides were interpreted by a pathologist (S.O.) blinded to any other clinical or laboratory data. The results of the p53 assays on these tumors have been previously correlated with other molecular features in tumors.24Ogino S. Brahmandam M. Kawasaki T. et al.Combined analysis of COX-2 and p53 expressions reveals synergistic inverse correlations with microsatellite instability and CpG island methylator phenotype in colorectal cancer.Neoplasia. 2006; 8: 458-464Abstract Full Text PDF PubMed Scopus (74) Google Scholar, 26Ogino S. Kawasaki T. Kirkner G.J. et al.Loss of nuclear p27 (CDKN1B/KIP1) in colorectal cancer is correlated with microsatellite instability and CIMP.Mod Pathol. 2007; 20: 15-22Crossref PubMed Scopus (52) Google Scholar, 28Ogino S. Brahmandam M. Cantor M. et al.Distinct molecular features of colorectal carcinoma with signet ring cell component and colorectal carcinoma with mucinous component.Mod Pathol. 2006; 19: 59-68Crossref PubMed Scopus (206) Google Scholar Moreover, the categorization we used in this report has previously been shown to correlate sufficiently well with TP53 sequencing analyses (sensitivity, 0.85; specificity, 0.77 with a chosen cut point of 50% or more positive cells).29Curtin K. Slattery M.L. Holubkov R. et al.p53 alterations in colon tumors: a comparison of SSCP/sequencing and immunohistochemistry.Appl Immunohistochem Mol Morphol. 2004; 12: 380-386Crossref PubMed Scopus (27) Google Scholar, 30de Jong K.P. Gouw A.S. Peeters P.M. et al.P53 mutation analysis of colorectal liver metastases: relation to actual survival, angiogenic status, and p53 overexpression.Clin Cancer Res. 2005; 11: 4067-4073Crossref PubMed Scopus (30) Google Scholar We excluded women who did not complete the baseline 1980 dietary questionnaire or recorded implausible dietary data (n = 29,279); reported a baseline history of cancer (except nonmelanoma skin cancer; n = 3627), inflammatory bowel disease, hereditary nonpolyposis colon cancer, or a familial polyposis syndrome (n = 103); or died before baseline (n = 1). After these exclusions, 88,691 women were eligible for analysis and accrued follow-up time beginning on the month of return of their baseline questionnaire and ending on the month of diagnosis of colon cancer, death from other causes, or June 2002, whichever came first. In a previous analysis of this cohort, folate intake was significantly associated with the risk of colon cancer but had no influence on the risk of rectal cancer14Giovannucci E. Stampfer M.J. Colditz G.A. et al.Multivitamin use, folate, and colon cancer in women in the Nurses' Health Study.Ann Intern Med. 1998; 129: 517-524Crossref PubMed Scopus (668) Google Scholar; as a result, we did not consider incident rectal cancer among the study participants in this analysis. Like rectal cancer cases, cases of colon cancer for which we were unable to assay tumoral p53 expression were censored from the analyses at their date of diagnosis and were not included as end points. We calculated incidence rates of colon cancer for participants in a specific category of folate intake by dividing the number of incident cases by the number of person-years. We computed relative risks (RR) by dividing the incidence rate in one category by the incidence rate in the reference category and used Cox proportional hazards modeling to control for multiple variables simultaneously and to compute 95% confidence intervals (CIs). With the exception of folate, vitamin B6, methionine, and alcohol, for which we used baseline information in our primary analyses, we used the most updated information for all covariates before each 2-year interval. In secondary analyses, as previously described,31Hu F.B. Stampfer M.J. Rimm E. et al.Dietary fat and coronary heart disease: a comparison of approaches for adjusting for total energy intake and modeling repeated dietary measurements.Am J Epidemiol. 1999; 149: 531-540Crossref PubMed Scopus (882) Google Scholar to reduce in-person variation and to estimate long-term intake better, we also used the cumulative average intake of folate as reported on all available questionnaires up to the start of each 2-year follow-up interval. In subanalyses, we evaluated the risk of colon cancer by p53 expression status among women low both in folate and vitamin B6 intake. To examine a potential effect of folate on preneoplastic lesions, we further conducted analyses restricting to colon tumors that occurred within 5 years after baseline nutrient assessment. To compare the specific effect of intake of folate and other nutrients on colon cancer risk according to p53 expression status, we used a previously described method of competing risk analysis utilizing duplication method Cox regression.32Lunn M. McNeil D. Applying Cox regression to competing risks.Biometrics. 1995; 51: 524-532Crossref PubMed Scopus (771) Google Scholar, 33Glynn R.J. Rosner B. Comparison of risk factors for the competing risks of coronary heart disease, stroke, and venous thromboembolism.Am J Epidemiol. 2005; 162: 975-982Crossref PubMed Scopus (316) Google Scholar This methodology permits estimation of separate regression coefficients for nutrient intake stratified by the type of outcome (eg, p53-overexpressing cancer vs p53 wild-type cancer). We assessed the statistical significance of the interaction between the risk estimates according to tumor type using a likelihood ratio test that compared the model that allowed for separate associations of folate and other nutrients according to p53 expression status with a model that assumed a common association. We examined the possibly nonlinear relation between folate, vitamin B6, and the RR of colon cancer by p53 expression status nonparametrically with restricted cubic splines.34Durrleman S. Simon R. Flexible regression models with cubic splines.Stat Med. 1989; 8: 551-561Crossref PubMed Scopus (2030) Google Scholar Tests for nonlinearity used the likelihood ratio test, comparing the model with only the linear term with the model with the linear and the cubic spline terms. Restricted cubic spline functions were plotted with 4 knots (3 df). We used SAS version 9.1.3 (SAS Institute, Inc, Cary, NC) for all analyses. All P values are 2 sided. Among 88,691 women included in these analyses, those with a baseline folate intake <200 μg/day were slightly younger and more likely to smoke and to be sedentary compared with women with folate intake ≥400 μg/day (Table 1). They were also less likely to use aspirin or postmenopausal hormones regularly. In addition, only 8% of women with folate intake <200 μg/day used multivitamins, whereas among those with folate intake ≥400 μg/day, 84% reported current multivitamin use.Table 1Baseline Characteristics of the Nurses' Health Study CohortEnergy-adjusted folate intake (μg/day)<200200–299300–399≥400(n = 20,907)(n = 28,882)(n = 12,997)(n = 25,905)Dietary intakeaNutrient values (folate, vitamin B6, vitamin B12, methionine, and calcium) represent the mean of energy-adjusted intake. Folate (μg/day)159246341678 Beef, pork, or lamb as a main dish (servings/wk)3.12.62.32.3 Vitamin B6 (mg/day)1.592.052.765.15 Vitamin B12 (mg/day)5.556.457.7815.1 Alcohol (g/day)6.76.46.06.3 Methionine (mg/day)1.741.861.951.93 Calcium (mg/day)607723798812Other characteristics Median age (y)45.546.847.547.0 Former or current smoker (%)60565455 Pack-yearsbPack-years were calculated for former and current smokers only.23.320.418.719.2 Regular aspirin user (%)31323235 Body mass index (kg/m2)24.424.524.324.0 Physical activity, METS/wk (%)cMETS are metabolic equivalents. This was calculated based on the frequency of a range of physical activities (such as jogging) in 1986.11.113.815.815.6 Postmenopausal (%)dHormones are defined as postmenopausal estrogen or estrogen/progesterone preparations. Percent of never, past, and current use was calculated among postmenopausal women only.44444444 Never used hormones64626159 Past use of hormones18191919 Current use of hormones18192022 Current multivitamin use (%)8132484 Prior lower endoscopy (%)2222 Colorectal cancer in a parent or sibling (%)8878NOTE. Dietary intake and other characteristics at baseline questionnaire in 1980 (mean value unless otherwise indicated). All values have been directly standardized according to the age distribution of the cohort.a Nutrient values (folate, vitamin B6, vitamin B12, methionine, and calcium) represent the mean of energy-adjusted intake.b Pack-years were calculated for former and current smokers only.c METS are metabolic equivalents. This was calculated based on the frequency of a range of physical activities (such as jogging) in 1986.d Hormones are defined as postmenopausal estrogen or estrogen/progesterone preparations. Percent of never, past, and current use was calculated among postmenopausal women only. Open table in a new tab NOTE. Dietary intake and other characteristics at baseline questionnaire in 1980 (mean value unless otherwise indicated). All values have been directly standardized according to the age distribution of the cohort. We documented 399 incident cases of colon cancer accessible for p53 expression data during 1,861,916 person-years. Using immunohistochemical analysis of these 399 colon cancers in our tissue microarrays, 143 tumors (35.8%) were found to be p53 overexpressing. As in our previous studies,14Giovannucci E. Stampfer M.J. Colditz G.A. et al.Multivitamin use, folate, and colon cancer in women in the Nurses' Health Study.Ann Intern Med. 1998; 129: 517-524Crossref PubMed Scopus (668) Google Scholar, 35Wei E.K. Giovannucci E. Selhub J. et al.Plasma vitamin B6 and the risk of colorectal cancer and adenoma in women.J Natl Cancer Inst. 2005; 97: 684-692Crossref PubMed Scopus (93) Google Scholar, 36Fuchs C.S. Willett W.C. Colditz G.A. et al.The influence of folate and multivitamin use on the familial risk of colon cancer in women.Cancer Epidemiol Biomarkers Prev. 2002; 11: 227-234PubMed