We found that traditional HP from group I patients had very little methylation of MINTs or genes. In contrast, group II patients, clinically fulfilling the criteria of hyperplastic polyposis, were heavily methylated both at MINTs 2, 12, and 31 and for genes, HPP1, p14ARF, and p16INK4a. In particular, MINTs were very discriminating between the two groups. When MP were included in group II HP, MINT1 methylation was significantly different from group I HP lesions. This could indicate that MINT1 is methylated at a later stage than other MINTs in HP in hyperplastic polyposis.
The decision to collect lesions presenting in two contrasting clinical scenarios served to highlight the molecular differences across the various classes of lesion and excludes a single linear model of accumulating genetic alterations. Serrated lesions and polyps of the colorectum, encompassing ACF, HP, MP, and SA, are unlikely to represent different stages of a single pathway of morphogenesis. ACF39
and HP are common lesions compared with MP and SA,4
indicating that the majority do not progress along the “serrated pathway”.
Classification of “serrated polyps” has become complex and somewhat controversial in recent years and cannot be fully resolved in the present study. When SA were first highlighted it was appreciated that some resembled HP whereas others had a more adenomatous appearance.4
Lesions termed HP variant in this study were conceived as occupying the HP end of the serrated spectrum yet did show features that have been associated with adenomas. On this basis and in order to address the possibility that these lesions are precancerous, others have introduced the term “sessile serrated adenoma”.27–29
The recent recognition that serrated polyps share two molecular alterations, namely CIMP-high status and mutation of BRAF
, with sporadic MSI-H CRC, strengthens the concept of a serrated pathway of colorectal tumorigenesis.40–42
The flow diagram illustrates our concept of the early stages of the serrated neoplasia pathway leading to MSI-H CRC (fig 4). Serrated lesions with K-ras
mutations include ACF and traditional HP but these have little or no potential for neoplastic evolution. There are two pathways to an SA or MP, both implicating CIMP-high status, mutation of BRAF
, and loss of function of the DNA repair genes MGMT
that are inactivated by promoter hypermethylation. There may be another pathway via HP variants that are CIMP-high but have a K-ras
mutation instead of BRAF
, and MGMT
inactivated rather than hMLH1
. We have published evidence for this group of MSI-L/MSS CRCs.22
Figure 4 Aberrant crypt foci (ACF) and hyperplastic polyps (HP) are conceived as non-progressing lesions despite the frequent finding of K-ras mutations. The genetic alterations underlying the HP variant implicate the same signalling (more ...)
There were no differences in the frequency of K-ras
mutations across groups I and II. However, all K-ras
mutations in group II polyps were restricted to a single subject (II.1). This patient differed from other subjects with hyperplastic polyposis in multiple respects. She did not have cancer and HP were more numerous and showed less methylation of MINTs (although more methylation than in HP from group I subjects). Lack of molecular homogeneity among polyps from different subjects with hyperplastic polyposis has been noted previously.26
This study also found evidence of molecular heterogeneity among group I subjects. Patient I.7 differed from other group I subjects in having a CIMP-high cancer of the distal colon with MSI-L (instability in MYCL), a CIMP-high SA, and the only example of a HP variant among group I subjects that was also CIMP-high. While falling short of the clinical definition of hyperplastic polyposis,31
the molecular features were unlike those of group I lesions and similar to those of group II lesions. This suggests that hyperplastic polyposis may exist in less penetrant forms that are nevertheless clinically important in terms of cancer risk. Additionally, this case illustrates the fact that lesions with extensive DNA methylation are not restricted to the proximal colon.
Patients with MSI-H cancers frequently have HP but in insufficient numbers to warrant a diagnosis of hyperplastic polyposis. In addition, the HP variant may occur sporadically, outside the condition hyperplastic polyposis.28
Therefore, hyperplastic polyposis, while rare, may represent the most severe manifestation of a predisposition to produce large serrated polyps with extensive DNA methylation and having an increased risk of malignancy.
Dysregulation of methylation seems to act at multiple steps to allow the cell to escape from cell growth control and apoptosis. CIMP is an effective mechanism for gene silencing that does not involve mutations, but none the less produces a stable epigenetic state that is transmitted to daughter cells in a clonal fashion. The concept that hypermethylation occurs on the basis of a pathological lesion has been disputed recently43
but we have shown here that a distinct lesion presenting in hyperplastic polyposis patients exhibits aberrant control of methylation of many genes, including key tumour suppressor genes.
The lack of K-ras
mutation in CIMP-high polyps with methylation of hMLH1
was mirrored by the low frequency of K-ras
mutations in sporadic MSI-H cancers.11
The latter showed frequent CIMP-high status that included hMLH1
methylation and have been related histogenetically with HP.23,40,44
None of the cancers in this study were MSI-H or showed hMLH1
methylation, even in subjects with hyperplastic polyposis. It is known that cancers in hyperplastic polyposis may be MSI-H, MSI-L, or MSS.23
The common denominator in cancers complicating hyperplastic polyposis may therefore not be the mutator phenotype but a predisposition to DNA methylation that may or may not implicate particular DNA repair genes, such as hMLH1
. In this study, all samples of carcinoma in situ or invasive malignancy from patients with hyperplastic polyposis showed at least CIMP-low status and the minimum number of MINTs or genes found to be methylated in this group was three (fig 3). Four of six cancers occurring in group I subjects were MINT-neg and a fifth may have been incorrectly assigned to group I (see above).
Nor is extensive DNA methylation explained merely on the basis of anatomical location. Left sided HP from group II subjects showed more extensive DNA methylation than group I HP. Additionally, it has been pointed out that whereas traditional HP are restricted to the distal colorectum, the HP variant may occur distally as well as proximally.29
It may therefore not be possible to distinguish clinically significant from non-significant serrated polyps on the basis of the number of polyps or anatomical location.
In summary, the findings lend support to the concept of a serrated pathway to CRC in which DNA methylation plays an important role. However, only a subset of serrated polyps is implicated. Among these is a group that is well represented in subjects with hyperplastic polyposis, is characterised by CIMP-high status, methylation of hMLH1
, absence of K-ras
mutation, and a high frequency of dysplasia. At the morphological level, most of these lesions are equivalent to the recently described “sessile serrated adenomas” or to mixed polyps when dysplastic subclones are present. It is hypothesised that these are the precursors of sporadic MSI-H CRC. Similar lesions but lacking methylation of hMLH1
and having a K-ras
mutation may be precursors of CIMP-high CRC that are MSS or MSI-L. ACF and classical HP differ from these lesions in their predilection for the distal colorectum, lower frequency of DNA methylation, and higher frequency of K-ras
mutations. The pattern and extent of DNA methylation may determine whether a lesion remains small and clinically insignificant or has the potential for progression. Interpatient differences among subjects with hyperplastic polyposis indicate subtle mechanistic differences that counter the suggestion that DNA methylation occurs exclusively as a stochastic age related process.21
The finding of familial clusters of hyperplastic polyposis26
points to an underlying genetic predisposition to extensive and early onset of DNA methylation.