The role of folate in colorectal carcinogenesis is complex and likely involves multiple metabolic pathways. These would include the role of folate in conversion of deoxy uracil monophosphate (dUMP) to deoxy thymidine monophosphate (dTMP). Failure to produce adequate supplies of dTMP results in uracil misincorporation, which has been linked to DNA strand breaks, loss of heterozygosity, and other forms of DNA instability. [2]

Additionally, folate is the intermediary carrier molecule for the labile methyl group in S-adenosylmethionine, which is responsible for nearly all methylation reactions in the body, including methylation of DNA. A minimum level of genomic methylation may protect against carcinogenesis as methylation is thought to stabilize the genome. [2, 3] Prior studies have shown that genomic methylation is typically lower in colon tumor tissue than in adjacent normal tissue in the same patient [4] and that genomic methylation in lymphocyte DNA is lower in subjects with adenoma compared to those without. [5] A second role of methylation in carcinogenesis is in regulation of gene activity through methylation in the promoter regions of genes essential to regulation of cell growth and replication. Aberrant methylation of these promoter regions can thus result in disregulation of the controls on cell growth and replication with obvious implications for carcinogenesis.

The classic Vogelstein model of a sequence of genetic alterations from APC mutations to p53 mutations that mark the transition from normal colonic epithelium to adenomatous polyp and then to invasive colorectal cancer describes about two thirds of colorectal cancers, and these tend to be more prevalent in the distal colon. [6, 7] A separate class of colon cancers comprises tumors displaying the CpG island methylator phenotype (CIMP). [8, 9] CIMP cancers are characterized by promoter methylation and hence gene silencing at multiple genes involved in carcinogenesis. CIMP cancers account for 10 to 30% of all colorectal cancers and are concentrated in the proximal colon. Interestingly, CIMP cancers appear to arise not through the classic Vogelstein, APC mutation-based tubular adenoma precursor lesion but rather through either a villous or serrated adenoma or a hyperplastic polyp precursor lesion. [10, 11, 12]

The concept that is explored in this study—whether inadequate folate availability in the colon leads to inappropriate hypermethylation of several important tumor suppressor genes--is biologically plausible and supported by work in a pre-clinical model, where folate inadequacy was observed to lead to promoter hypermethylation and reduced expression of a critical tumor suppressor gene. [13] However, the issue of whether the same phenomenon operates in the human colon has not been adequately addressed, and there is little consensus among the few observational studies that have tried to examine the question. [14, 15, 16] Perhaps the conflicting results relate, in part, to the fact that they have each used blood folate levels, or dietary intake, as a proxy measure of colonic folate status.

Among the 813 women with tissue folate information available in this study, 170 had at least one prevalent adenomatous polyp, and 37 of these women had at least one advanced adenoma. We observed a modest, non-significant inverse association between tissue folate concentration and prevalent adenomas overall (OR = 0.68, 95% CI 0.40–1.16 for Q5 vs. Ql; p trend = 0.53). However for advanced adenomas, the most clinically relevant outcome other than invasive colorectal cancer, women in the top quintile of tissue folate concentration had a statistically significant lower risk (OR = 0.24, 95% CI 0.06–0.93; p trend = 0.049).

Analyses by anatomic location indicate a subsite specificity for the association between tissue folate concentration and adenomas (Table 3). In the proximal colon the OR for Q5 vs. Q1 was 0.56 (although the 95% CI did include 1.0), and furthermore, in Q3 and Q4, the point estimates, 0.48 for Q3 and 0.49 for Q4, were statistically distinct from the null. By contrast, there was no evidence of association between tissue folate concentration and prevalent adenomas in the distal colon. These results suggest that tissue folate may be important in the etiology of adenomas that arise in the proximal colon, but not in those arising in the distal colon.

The etiologic mechanisms underlying the basis for the three primary molecular subtypes of colorectal cancer remain incompletely understood, though it is clear that methylation of specific genes (e.g., hMLH1), of the genome generally (i.e., genomic methylation), and of classes of genes (i.e., methylation profiles) are all involved. In particular, the CIMP cancers are characterized by aberrant methylation of promoter regions in multiple genes related to regulation of cell growth and differentiation. Although it is important to emphasize that they do not prove an association between tissue folate concentration and a specific molecular subtype of colorectal cancer, our findings from women enrolled in the CONCeRN study do suggest that folate status in the colonic epithelial tissue may be linked to the aberrant methylation patterns that can result in cancers developing from one of these three molecular pathways, the CIMP pathway.

Prior research in this area has not adequately exploited concentrations of folate in normal colonic biopsies to examine the potential etiologic role of folate in colorectal carcinogenesis. Our study was distinct in that we were able to measure folate status directly in the target tissue of interest, colonic mucosa, whereas nearly all previous research in humans has relied on markers of folate status (i.e., dietary intake or blood concentration) that were in effect proxy measures of folate concentration in the colon.

Our design also allowed us to use tissue obtained from asymptomatic, average-risk women referred for standard colorectal cancer screening meaning we were not reliant on a small case series as previous studies of tissue folate concentration have been. [24, 25, 26] Furthermore, the colonoscopic procedure ensured that we were able to identify polyps at all anatomic locations in the colon and rectum, a critical design feature given the different anatomic distribution of the various molecular subtypes of colorectal cancer. In combination, the availability of direct measures for tissue folate concentration, the enrolment of 813 asymptomatic women, and the identification of neoplasia at all anatomic subsites via complete colonoscopy (resulting in the classification of women as having healthy colons, adenomatous polyps, and/or hyperplastic polyps) provided a robust and unique resource to study this question.

The value of our effort to focus on the association of folate status with one specific molecular subtype of colorectal neoplasia, the CIMP subtype, is apparent when we contrast our results to those from one of the few prior studies using human tissue specimens. Meenan and colleagues found that folate concentration in adjacent normal tissue in subjects with adenomas or cancer was similar to that in normal epithelial tissue from subjects with healthy colons, [24] a result that would appear to be in contradiction to our findings. All case subjects in the earlier analysis, however, had either distal cancer or distal adenomas, and, as our analysis suggests, the association in the distal colon would be expected to be null, just as they observed. In contrast, we were able to consider proximal location and to include hyperplastic polyps (both relevant to CIMP-related cancers) in defining our outcomes thus allowing us some ability to isolate the CIMP-related polyps. After doing so, we were able to observe a significant association that was not found if we included polyps of histologic type or anatomic location that made them unlikely to be CIMP-related.

A recent report from Levine and colleagues adds additional evidence in support of a site-specific and molecular subtype-specific effect of folate and methylation on colorectal cancer etiology. [27] In a large family-based case-control study, the MTHFR 677 TT genotype was associated with a decreased risk of MSI-S/MSI-L tumors but an increased risk of MSI-H tumors, and it was further associated with decreased risk of distal and rectal tumors but an increased risk of proximal tumors. Together with our results, these findings provide evidence that the effects of folate metabolism on colorectal carcinogenesis are specific in nature to the type of tumor involved. The effects of folate metabolism and folate status on proximal, microsatellite-unstable tumors are quite distinct from the effects on distal tumors.

The wide range of colonic folate concentrations that were observed is perhaps of importance. The mean concentration of colonic folate among those in the highest quintile was six times greater than that observed in the lowest quintile (range: 0.273 – 1.676 µg/g for mean of Q1 to mean of Q5). Moreover, this wide range of concentrations was present even though the total mean folate intake of two extreme quintiles only differed by ~40%. Thus, considerable biological variability in colonic folate concentrations exists within a relatively healthy, ambulatory single-gender population and the extent of variability extends far beyond the variation in habitual intake of the vitamin, suggesting other factors play important roles in determining colonic folate levels. If mucosal folate levels are indeed a causal factor in determining the risk of neoplastic transformation, such a pronounced variation between individuals makes it easier to understand why some individuals would be at higher risk than others due to this factor.

It is estimated that far less than 10% of adenomas will ever go on to develop into cancers, and therefore, the recognition of the ‘advanced adenoma’ as a lesion that is much more likely to progress to cancer [28, 29] was an important one since it is therefore likely to be a more accurate biomarker of cancer risk. Consequently it is of considerable interest that colonic folate concentrations were observed to be significantly associated (in an inverse fashion) with advanced adenomas, but not with other types. This suggests that colonic folate concentrations are more closely linked with those lesions that are most likely to progress to cancer than with those that are most likely to remain indolent and benign.

An important limitation to our analysis is the lack of specific molecular subtype information for our outcomes. Our use of a proxy variable (what we described as lesions most likely to be CIMP-related rather lesions with clinically-determined CIMP pathology) depends on an assumption that CIMP and non-CIMP tumors, and their precursor lesions, have an anatomic specificity for proximal and distal location, respectively. This is clearly an oversimplification. In truth, the low levels of folate could be associated with the non-CIMP tumors that develop in the right colon from advanced adenomas. Furthermore, not all methylation that occurs in colorectal neoplasms is CIMP-related. However, the available evidence suggests this is not likely to be an oversimplification that yields inappropriate conclusions. Using prospectively collected specimens from a hospital-based frozen tumor bank containing 879 colorectal cancer cases, Whitehall and colleagues [30] found that 88% of cancers classified as having a high degree of CIMP characteristics and that were also MSI-H occurred in the proximal colon. Among those cancers that were CIMP high but were not MSI-H, 50% were in the proximal colon. By contrast, only 17% of non-CIMP cancers occurred in the proximal colon. With respect to hyperplastic polyps, a recent paper from Vaughn and colleagues [31] reported that while BRAF mutations (hypothesized to be the initiating mutation for the hyperplastic polyp-CIMP tumor pathway [32]) were very common in both proximal and distal hyperplastic polyps, 48% of proximal hyperplastic polyps had CIMP characteristics in contrast to distal hyperplastic polyps where only 4% did so. Thus while the classification we used is certainly imperfect, it is clear that the vast majority of CIMP tumors arise in the proximal colon, and that a large fraction, though admittedly not all, of the lesions in the proximal colon are CIMP-related (i.e., lesions that arise from the same molecular pathway that will produce CIMP cancers).

Nonetheless, what we describe as likely to be CIMP-related polyps are in fact proxies for the true outcome of interest, and hence they necessarily involve some degree of misclassification. While it is impossible to quantify the degree of misclassification, the most likely result of this misclassification would be to bias the results toward the null. Yet despite this, we were still able to observe an inverse association between tissue concentration of folate and prevalence of polyps crudely classified as most likely to be CIMP-related. Certainly a more-precise and more-direct determination of CIMP status would be a design feature that future investigations would do well to employ, but unfortunately this was not available for the CONCeRN study.

The analysis we conducted used tissue biopsies obtained from the region of the splenic flexure, but the polyps we found were distributed across the entire length of the colon and rectum. In other words, the normal tissue biopsies in which we measured folate concentration were not necessarily adjacent to the polyps that defined our endpoints. In fact, the site where the association of polyps with the tissue folate concentration was strongest, the proximal colon, was by definition distant from the location of the tissue biopsies. This suggests that the association we observed was the result a generalized field effect, that low concentration of folate in the normal mucosa of the colon in general predisposed the proximal colon to risk of adenoma or hyperplastic polyp.

One complication to this conclusion of a field effect is the cross-sectional nature of the study design. Given that tissue folate concentration was assessed at the same time as the polyps were identified, it is impossible to determine conclusively which preceded the other. That said, it seems unlikely that these small lesions in asymptomatic women at average risk of colorectal cancer could produce changes in folate concentration in normal tissue elsewhere in the colon.

Finally, results from some recent supplement trials have raised concern about the possibility of a paradoxical cancer-promoting effect of high-dose folate, and that issue may have relevance in the present study. [33] We considered this possibility in our analyses but found no evidence that such an effect was present in this study population (data not shown). Its absence in our dataset was, in retrospect, not surprising since more than 95% of the subjects were consuming less than 940 µg of folate per day. Given that the paradoxical cancer-promoting effect has been observed only in people who are receiving exceptionally high amounts of folic acid on a habitual basis, the level of intake in the CONCeRN Study population is likely far below the threshold at which we might expect to observe this cancer-promoting effect.