The overall low frequency of cancers with one or more APC mutation (22%) is explained in part by the over representation of sporadic MSI-H and HNPCC cases in this series and in part by limiting the search for APC mutation to the hot spot MCR in exon 15. It could be argued that the low frequency of APC mutation (4.8%) in sporadic MSI-H results from the fact that APC mutation occurs outside the MCR in this subset. This interpretation is unlikely for several reasons. The observation fits with more extensive studies of APC mutation in colorectal cancers, in which sporadic MSI-H cancer was carefully distinguished from HNPCC.21,25
The finding also fits with the frequent retention of a normal β catenin immunolocalisation pattern in this subset.23,24
The frequency of APC mutation in HNPCC cancer is also low (26.7%). However, within this subgroup, disruption of the WNT signalling pathway may also occur through mutation and oncogenic activation of β catenin.22
Aberrant immunolocalisation of β catenin within HNPCC cancers29
fits with the mutational evidence for WNT pathway disruption.
“These findings indicate that budding is a dynamic process under genetic control and not merely the result of architectural disruption caused by a host immune reaction at the tumour margin”
The frequency of APC mutation in the MSI-L group (32.6%) differed from the MSI-H group (4.8%), but not from MSS (37.5%) cancers. Although the difference between MSI-L and MSS cancers was non-significant, the trend across the cancer subgroups fits with findings for other markers of WNT pathway disruption—specifically, loss of heterozygosity at 5q and aberrant immunolocalisation of β catenin.23
Contamination of the MSI-L group by MSI-H cases is an unlikely explanation for the low frequency of APC mutation in the MSI-L group or the intermediate findings (between MSI-H and MSS cancers) with respect to tumour budding. None of the MSI-L cancers in our study showed either instability at mononucleotide markers or loss of expression of DNA mismatch repair proteins (data not shown). Furthermore, we have shown previously that MSI-L cancers are distinguished from MSI-H cancers by a high frequency of methylation of the DNA repair gene MGMT and a high frequency of K-ras mutation.30
Methylation of MGMT has been associated with G : C to A : T transitions in K-ras and TP53.27,28
Although most of the MSI-L cancers in our study showed methylation of MGMT,30
50% of APC mutations were deletions on short polyadenine tracts and only one of 14 truncating mutations was a G : C to A : T transition. This suggests that in a subset of MSI-L neoplasms, methylation of MGMT may be acquired in adenomas subsequent to early APC mutation. Large selection pressures must account for the frequent finding of frameshift APC mutations in tumours that are not otherwise prone to instability in mononucleotide tracts.
The frequencies of tumour budding and of APC mutation were distributed in similar patterns across the four subsets of colorectal cancer (table 2). Within the subsets, however, there was no correlation between budding and APC mutation. In the case of MSI-H and HNPCC cancers, both features were uncommon. Within the MSI-L and MSS groups were cancers with budding but no APC mutation, and vice versa. Combining these groups, budding occurred in 20 of 39 cases without mutation and seven of 20 cases with mutation. However, it is inevitable that the frequency of APC mutation is underestimated in both groups. In addition, the frequency of budding in both groups was high compared with other studies and may have been overestimated. For example, the frequency of budding in a series of rectal cancers (which would have been unlikely to have included many sporadic MSI-H or HNPCC cancers) was around 30%.5
A further explanation for the discordant findings may be the prerequisite for factors other than a single APC mutation (for example loss of heterozygosity at 5q) or a fully independent mechanism (see below). Budding was present in the single cancer (an HNPCC cancer) with two (presumably biallelic) truncating mutations.
In our study, a subset of cancers was immunostained for β catenin and p16. As has been shown by others, the invasive margin and specifically tumour buds showed increased expression of β catenin and p16.7,8
The increased expression of β catenin was both cytoplasmic and nuclear, whereas p16 expression was mainly cytoplasmic. These findings indicate that budding is a dynamic process under genetic control and not merely the result of architectural disruption caused by a host immune reaction at the tumour margin. Tumour budding is known to be associated with reduced proliferation, yet nuclear cyclin D1 expression is increased.8
The increased cytoplasmic p16 expression may explain this paradox. For example, p16 may bind cytoplasmic cyclin dependent kinase 4 (cdk4) and block its nuclear translocation. Cyclin D1 is known to form relatively inert complexes with cdk2 and would therefore, in the absence of nuclear cdk4, compete with cyclins A and E (fig 4).31
Although the preceding model is speculative and depends upon the demonstration of cytoplasmic binding of cdk4 by p16, it provides an economical explanation for the discordant findings of raised p16, raised cyclin D1, and lowered proliferation at the invasive margin of colorectal cancer.8
It is possible that the expression of p16INK4a
and WNT pathway disruption may serve as joint prerequisites for tumour budding. The lack of tumour budding in MSI-H cancers would be explained by the known silencing of p16INK4a
through methylation of the promoter region of the gene,32
or by the lack of WNT pathway disruption (or both). There was no correlation between the methylation of p16INK4a
and budding among the non-MSI-H cancers in our study (data not shown).
Figure 4 Dysregulated p16 binds cyclin dependent kinase 4 (cdk4), which results in cytoplasmic sequestration of cdk4. Binding between nuclear cyclin D1 and cdk2 results in an inert complex but serves to compete with the binding of cdk2 to cyclins A and E. This (more ...)
In summary, the feature of tumour budding has been associated with invasion, metastasis, and poor prognosis. The findings of our present study suggest that the lack of this feature in sporadic MSI-H colorectal cancer, in turn explained by an intact WNT signalling pathway and/or methylation of p16INK4a , may serve as the explanation for the low biological aggressiveness of this subgroup.
Take home messages
- Tumour budding is associated with invasion, metastasis, and an adverse prognosis
- The lack of budding in microsatellite instability high colorectal cancer may account for the improved prognosis of this subset and may be explained by an intact WNT signalling pathway and/or inactivated p16INK4a