In this cohort of patients with colon cancer, we examined the effect of the CpG island methylator phenotype (CIMP), microsatellite instability (MSI), and BRAF
mutation on patient outcome. CIMP-high status was independently associated with a low cancer-specific mortality, whereas BRAF
mutation was associated with a significant increase in cancer-specific mortality. Consistent with other studies,[20
] we found that MSI-high tumors showed a trend towards an association with longer survival. Of note, the adverse effect of BRAF
mutation appeared to be limited to tumors that were not CIMP-high. Although our observations need to be confirmed by other independent studies, the associations of CIMP and BRAF
mutation with clinical outcome were consistent across the two independent prospective cohort studies in this analysis.
The relationship between CIMP, MSI, and BRAF
mutations in colon cancer is complex. In our cohort, 70% of BRAF
-mutated tumors exhibited CIMP-high, and 70% of CIMP-high tumors exhibited MSI-high. Among patients who do not manifest hereditary nonpolyposis colorectal cancer (HNPCC), MSI-high is often the consequence of promoter methylation (and subsequent silencing) of MLH1
, a DNA mismatch repair gene.[8
] In fact, CIMP and BRAF
tests are used to exclude HNPCC among patients who exhibit MSI-high, since HNPCC seldom exhibits CIMP or BRAF
Studying epigenetic and/or genetic alterations is increasingly important in cancer research.[3
] To decipher the apparently complex effect of CIMP and BRAF
mutation on patient survival, we utilized a validated expanded panel of 8 methylation markers for CIMP diagnosis in colorectal cancer.[8
] To determine DNA methylation status at each locus, we used a quantitative method that appears to reproducibly differentiate high-level from low-level methylation.[35
] Our validated criteria for CIMP-high are based on the bimodal distribution of tumors according to the number of methylated CIMP markers, and the observation that CIMP-high is associated with BRAF
mutation while CIMP-low is associated with KRAS
] Our large sample size from the two independent cohort studies enabled us to estimate the frequencies of specific molecular features (e.g., CIMP-high, etc.) and cancer death rates at the population level.
Although prognostic factors have been extensively investigated for colon cancer,[20
] previous studies of CIMP and survival in colon cancer have yielded somewhat inconsistent results.[22
] Some studies suggested an adverse effect of CIMP on survival of patients with MSS tumors.[23
] However, accumulating evidence has been suggested that MSI-high tumors are associated with good prognosis regardless of CIMP status,[22
] which is in agreement with our current study (). BRAF
mutation has been associated with worse survival in MSS tumors, but there was little prognostic value of CIMP in multivariate analysis.[22
] Our findings of good prognosis in CIMP-high tumors appear to differ from the data in the previous studies.[23
] These discrepant observations might have resulted from differences in patient cohorts, methylation markers, criteria for CIMP, and/or the variable inclusion of other potential confounders (such as BRAF
mutation) in multivariate analysis models. In particular, we have previously observed worse prognosis associated with CIMP-high tumors in stage IV colorectal cancer in small phase I/II clinical trials.[25
] The possible reasons for the discrepant results are as follows: 1) A selection bias in the small clinical trials with only 5 CIMP-high tumors might have caused this discrepancy. 2) Data in our previous study[25
] with only 5 CIMP-high tumors might simply be the result by chance in the setting of a small patient population. A p value by the log-rank test is calculated by the Mantel-Haenszel chi-square test that can offer a far more accurate p value with a large sample size. Thus, we would emphasize the importance of a large sample size in any clinical study. Because of the use of the expanded CIMP marker panel (including the 5 new markers described by Weisenberger et al.[8
]) in the current study, good prognosis might be specifically associated with CIMP-high tumors defined by these new CIMP makers. Our observations of good prognosis in CIMP-high tumors appeared to be consistent across all stages (I to IV), further supporting that CIMP-high tumor is a biologically indolent subtype. In addition, we found that, after jointly examining CIMP and BRAF
status, CIMP-high predicted a lower colon cancer-specific mortality (regardless of BRAF
status) compared to CIMP-low/0 BRAF
-mutated tumors, whereas the deleterious effect of BRAF
mutation was not as evident in patients with CIMP-high tumors.
In our cohorts, data on cancer treatment are limited. Nonetheless, it is unlikely that chemotherapy use differed according to tumoral CIMP, MSI or BRAF
status, especially since such data were not typically available to patients or treating physicians. It still remains a possibility that differential response to chemotherapy according to a specific molecular variable (e.g., MSI) might confound our findings. Further studies are necessary to examine whether response to chemotherapy may be differentially influenced by specific molecular features in colon cancer. In addition, beyond cause of mortality, data on cancer recurrences were not available in these cohorts. Nonetheless, given the median survival for metastatic colon cancer was approximately 10 to 12 months during much of the time period of this study,[49
] colon cancer-specific mortality should be a reasonable surrogate for cancer-specific outcomes.
Despite the apparent effects of CIMP, MSI, and BRAF mutation on colon cancer-specific mortality, the influence of these tumoral events on overall mortality was markedly attenuated, which may have reflected the inclusion of earlier stage (I and II) patients in our analysis. In fact, when we limited our analysis to patients with either stage III or IV cancer, we observed similar effects of CIMP on both cancer-specific and all-cause mortality. Moreover, when we jointly classified patients according to both CIMP and BRAF status, we observed similar trends for cancer-specific and overall mortality among the entire patient cohort ( and Figure 4).
In conclusion, this large prospective study of colon cancer patients suggests that CIMP-high is independently associated with a low cancer-specific mortality. While BRAF mutation is associated with worse survival, CIMP-high appears to eliminate the adverse effect of BRAF mutation. Finally, while our data confirm the extensive body of evidence supporting a better prognosis for patients with MSI-high tumors, the good prognosis associated with MSI-high was abrogated in the presence of a BRAF mutation. Our finding that CIMP-high is an independent predictor of cancer survival may have significant clinical implications, although it needs to be confirmed by additional independent studies. Future studies to validate our observations should consider a joint examination of CIMP, MSI and BRAF mutation to decipher the role of these molecular features in biological and clinical behavior of colon cancer.