In this study, we show that methylation biomarkers play a differential role in CRC recurrence and DFS by tumor location. Using gene methylation and mutation, we could cluster cases into three distinct groups. The high methylation group demonstrated a significant association with CIMP+, BRAF mutation and MSI-H. The intermediate methylation group showed a significant association with high frequency of KRAS mutation. These molecular features were almost the same as those that we previously reported30
in a different population of patients, demonstrating the reproducibility of this classification. Methylation related biomarkers influenced recurrence and DFS in resected stage III proximal colon cancer, but not in distal colon cancer. The study adds to the growing literature on differences between proximal and distal cancers, and suggests that these may have to be taken into account in management and clinical trials in this disease.
CIMP negative colon cancers are evenly distributed throughout the colon, but CIMP1 colon cancers are principally located in proximal colon.32
The cause of this difference is unknown. It may be that site-specific carcinogens or differences in the cell of origin may explain this variation in DNA methylation. Still, only about half of proximal cancers are CIMP1, and our data now suggest that DNA methylation may help in classification of these patients for prognostic purposes. A high risk for recurrence in stage III CRC may lead to more intensive surveillance and novel adjuvant therapy strategies. We have previously reported that the methylation status of several CIMP markers was associated with poor survival in stage IV CRC14
but it is not yet known whether this is modulated by site. One of the paradoxical findings on the effects of CIMP on survival is the fact that CIMP1 is also associated with MLH1 methylation, which results in MSI. MSI is generally a favorable prognostic factor in CRC.15, 16
MSI was relatively rare in the population of patients we studied, which explains why the dominant effect of CIMP was negative on DFS. In a small, pilot analysis, we did find that MLH1 methylated cases a good outcome, while CIMP1, MLH1 unmethylated cases had a strikingly high recurrence rate, regardless of BRAF mutation. It is interesting to consider why the two groups of CIMP1 cases (MLH1 methylated/unmethylated) would have such opposing consequences. An attractive hypothesis is that the poor prognosis is imparted by DNA hypermethylation of genes such as WNT5A that result in a more aggressive behavior (increased invasion for example). In turn, this invasive phenotype may be countered by induction of an immune response that is most pronounced in MSI positive cases.33
Here, we confirm a previous report on the prognostic impact of LINE1 methylation on outcome of CRC,10
but show that this is also limited to proximal cancers. Others have examined the effect of global DNA methylation on clinical outcomes in various cancers. In one study, the level of global DNA methylation was significantly lower in prostate cancer than in the normal prostate, but there was no difference according to recurrence.34
In ovarian cancer, the level of LINE-1 methylation was significantly lower than in normal tissue and there was a shortened survival in the low methylation group35
. The mechanisms by which low levels of LINE-1 methylation are associated with a poor outcome remain to be determined. Possibilities include association with genomic instability, or with activation of expression of selected genes.
It is interesting to consider why DNA methylation was associated with recurrences in proximal cancers but not in distal cancers. A simple possibility relates to the rarity of CIMP1 cases in distal cancer, which limits our power to detect a prognostic impact there. Larger studies should address this issue. It is also possible that some other molecular marker, not measured here, has a dominant effect on recurrences and thus negates the effect of methylation differences on outcomes. Indeed, deletions of chromosome 18 are associated with recurrences in stage III CRC,36
and these are more common in distal cancers and CIMP-negative cases.37, 38
Thus, it may be that DNA methylation is a dominant prognostic factor in proximal cancers, while genetic instability is a dominant prognostic factor in distal cancers. Our studies and these hypotheses, which need to be confirmed in a larger population, pave the way for individualized management of stage III CRC.
In summary, methylation biomarkers such as methylation of WNT5A, CIMP markers and LINE-1 can predict disease recurrence and DFS in resected, stage III proximal colon cancer, but not in distal cancers. However, as the number of CIMP1 cases was small in distal CRC, further study is required to validate our findings. Classification of CRC by both genetic and epigenetic profiles will likely improve the capability to predict prognosis and to apply tailored therapy in this disease, but the classification will also have to take into account differences between proximal and distal cancers.