A growing body of evidence supports the notion that at least three primary pathways to colorectal cancer exist, each with a unique molecular pathology, and each with distinct clinical features related to presentation, anatomic location, and prognosis. [11
] Risk factors for proximal colon cancers are distinct from those that arise in the distal colon. Distal cancers are more frequent in younger men while proximal cancers occur most commonly in older women [21
]. Familial adenomatous polyposis (germline mutation of the APC
gene) patients develop distal tumors, and hereditary non-polyposis colon cancer (germline mutation of mismatch repair genes, often MSH2
) patients develop proximal tumors [21
]. Of particular relevance to this study, long term use of folate-containing supplements was associated with reduced risk of proximal, but not distal, colon cancer in a cohort of U.S. nurses [22
]. Furthermore, in a case-control study from Australia, a polymorphism in the folate-dependent enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR
C677T) was associated with an increased risk of proximal colon cancer, especially among those with low folate intake [23
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
] 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
] 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.
In summary, we found that concentrations of folate in normal colonic mucosa were inversely though modestly associated with prevalent adenomatous polyps in asymptomatic women undergoing screening colonoscopy. Even more interestingly, if we defined the outcome to include those polyps most likely to be CIMP-related, and hence to polyps where aberrant methylation is most directly relevant, we also saw significant inverse associations with tissue concentrations of folate. Any other definitions of the outcome, definitions that included large numbers of polyps that were unlikely to be CIMP-related, resulted in null associations. These findings, though they are not definitive, are consistent with the hypothesis that risk factors for colorectal cancer may be specific to molecular subtype and in particular that folate status in colonic mucosa is an exposure that is etiologically important to CIMP cancers as distinct for other molecular subtypes.