A Prospective Study of Dietary Folate and Vitamin B and Colon Cancer According to Microsatellite Instability and KRAS Mutational Status

A high degree of microsatellite instability (MSI)-high due to defective mismatch repair is one of the mechanisms in colon carcinogenesis. Most sporadic MSI-high colon cancers are positively associated with MLH1 promoter methylation (1, 2) and inversely associated with KRAS mutation (3). Folic acid and related B vitamins (one-carbon nutrients) are essential for DNA methylation and nucleotide biosynthesis, and it is therefore plausible that chronic folate deficiency may be associated with MSI or KRAS mutation. Adequate dietary intake of these nutrients has previously been related to a lower colon cancer risk (4-6). However, whether their intake differentially affects molecular subtypes of colon cancer has not extensively been studied. We therefore assessed whether the influence of folate and B vitamin intake on colon cancer risk differed according to the presence of MSI-high or KRAS mutation in two prospective cohort studies where folate intake has been inversely associated with the risk of colon cancer (7, 8).

Two independent prospective cohort studies, the Nurses' Health Study (121,701 women followed since 1976; ref 9) and the Health Professional Follow-up Study (51,529 men followed since 1986; ref. 10), formed the study population. Information on potential risk factors and newly diagnosed cases of cancer was updated biennially. Dietary intake of various nutrients including folate, vitamin B₆, B₁₂, and methionine were assessed by self-administered semiquantitative food frequency questionnaires (11, 12). All nutrient contributions including those from supplements were added to the specific nutrient intake from foods to calculate a daily intake for each participant (11). We assumed an ethanol content of 13.1 grams for a 12-ounce (38-dl) can or bottle of beer, 11.0 grams for a 4-ounce (12-dl) glass of wine, and 14.0 grams for a standard portion of spirits.

All colon cancer cases were confirmed through medical record review by study physicians. We collected paraffin-embedded tissue blocks from hospitals where colon cancer patients underwent resections of primary tumors (10). Based on availability of adequate tissue specimens, we analyzed 669 colon cancers for MSI and KRAS. Characteristics of those for whom we did and did not analyze for molecular markers have previously been found to be very similar (10). Genomic DNA from paraffin-embedded tissue was extracted and KRAS codons 12 and 13 were sequenced as previously described (13). MSI status was determined using D2S123, D5S346, D17S250, BAT25, BAT26 (14), BAT40, D18S55, D18S56, D18S67, and D18S487 (i.e., 10-marker panel; ref. 15). MSI-high was defined as the presence of instability in ≥30% of the markers.

After excluding participants who did not complete the baseline dietary questionnaire, or reported a baseline history of cancer (except nonmelanoma skin cancer), inflammatory bowel disease, hereditary nonpolyposis colon cancer, or a familial polyposis syndrome, 88,691 women and 47,371 men were eligible for analysis. We used a previously described method of competing risk analysis using duplication method Cox regression to compare the specific effect of intake of folate and other nutrients on colon cancer risk according to MSI (or KRAS status; refs. 16, 17). We assessed the statistical significance of the difference 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 MSI (or KRAS status) with a model that assumed a common association. Established or suspected risk factors for colon cancer were included in the multivariate models. We used SAS version 9.1.3 for all analyses.

Among all 88,691 women and 47,371 men included in these analyses, those with a baseline folate intake of <200 μg/day were slightly more likely to eat meat, smoke and less likely to exercise or report multivitamin use (Table 1).

We documented 669 incident cases of colon cancer accessible for MSI or KRAS mutation data during 2,566,968 person-years. Of these, 127 (19%) tumors were MSI high and 242 (36%) tumors were KRAS mutated.

As in our previous studies (7, 8, 18, 19), we observed an inverse association between folate and vitamin B₆ intake and colon cancer risk among all cases in our cohort (Table 2). The multivariate relative risk of colon cancer was 0.75 [95% confidence interval (95% CI), 0.58-0.96] for a total folate intake of ≥400 μg, compared with <200 μg folate per day (Table 2). The influence of total folate intake did not differ according to MSI status; comparing extreme categories, the relative ratio (RR) was 0.79 (95% CI, 0.60-1.03) for microsatellite stable/MSI-low colon cancer and 0.61 (95% CI, 0.37-1.02) for MSI-high tumors (P_{heterogeneity} = 0.53). In contrast, the inverse relation between total folate intake seemed to be limited to KRAS wild-type cancer, although the tests for heterogeneity did not reach statistical significance (P_{heterogeneity} = 0.12). Results remained virtually unchanged when we limited our examination to cases that occurred before 1998 (before folate fortification became mandatory; data not shown).

We further examined the influence of intake of folate, vitamin B_6, B₁₂ and methionine (in quintiles) as well as alcohol intake, but the effects on cancer did not seem to differ by MSI or KRAS status (Supplementary Tables S1 and S2).

In this large prospective cohort study, we found that both low folate and vitamin B₆ intakes were associated with an increased risk of colon cancer, but these effects did not differ significantly by MSI or KRAS mutational status. Few studies have assessed the influence of one-carbon nutrients on colon cancer according to MSI or KRAS status and the results have been inconsistent (20-22). It is plausible that chronic folate deficiency may be associated with MSI or KRAS mutation, given the importance of folate in DNA methylation and synthesis. Martinez et al. (20) reported a lower incidence of KRAS-mutated colon adenomas in individuals with higher folate intake, but others have not found such an effect (21). Only one study has evaluated the association between dietary methyl donor intake and MSI and did not describe an important interaction (22). The absence of a significant association between one-carbon nutrients and MSI or KRAS mutational status in the current analysis suggests that more work is still needed to fully delineate the influence of one-carbon nutrients on colon carcinogenesis.

Our study has several important strengths. First, because we collected detailed, updated information on a number of dietary and life-style covariates relevant to colon carcinogenesis over up to 22 years of follow-up and with high follow-up rates, we were able to examine long-term exposures to one-carbon nutrients and to take into consideration important confounding factors. Second, our study is prospective, eliminating concerns about differential recall bias, particularly with regard to our dietary assessments. Any remaining bias from exposure misclassification would thus be nondifferential by nature, biasing our results toward the null.

Limitations of note relate to folate fortification, which became mandatory in 1998 (23). We did obtain multiple assessments of one-carbon nutrient intakes before fortification. In addition, because the development of colon cancer likely requires some induction period before the onset of a clinically apparent tumor, it is unlikely that the postfortification folate exposure would substantially influence colon cancer risk through 2002. Another potential limitation is that we were unable to obtain tumor tissue from all cases of confirmed colon cancer detected in the two cohorts. However, risk factors in cases unavailable for tissue analysis did not appreciably differ from those in cases with tumor tissue available.

In conclusion, our results show that the reduced risk of colon cancer associated with replete folate status does not seem to vary by MSI or KRAS mutational status. Additional studies are needed to elucidate the mechanisms underlying the preventive effect of one-carbon nutrients on colon cancer.

All of the authors declare no relevant conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the NIH. We thank the participants of the Nurses' Health Study and Health Professionals Follow-up Study for their cooperation and participation and hospitals and pathology departments throughout the United States for generously providing us with tumor tissue materials and Gregory Kirkner and Takako Kawasaki for technical assistance.