In this study utilizing a large sample size, we evaluated 16 methylation makers in an unbiased fashion. The 16 methylation markers included the 5 markers (CACNA1G
) that were selected by screening of 195 CpG islands 
and further validated to be included in the CIMP-high diagnostic panel (the above 5 plus CDKN2A
. By unsupervised hierarchical clustering analysis, the 5 methylation markers were clustered with each other as well as with MSI (microsatellite instability) and BRAF
mutation. Analysis of κ coefficient, sensitivity and specificity also demonstrated good performance of the 5 methylation markers with generally concordant methylation pattern. Utilizing the validated CIMP panel, we have deciphered the complex relations of CIMP-high with various clinical, pathologic and molecular features in colorectal cancer. Our data provide a rationale for the of the validated CIMP-specific methylation marker panel.
This study is the first extensive investigation to compare the 5 new CIMP-high markers 
with MINT1, MINT31 and other CpG islands, using a large sample size. Performance of the 5 new markers (CACNA1G
, and SOCS1
was consistently superior to that of WRN
, MINT1, CHFR
. MINT31, CDKN2A
, and p14 showed intermediate performance characteristics, and in hierarchical clustering analysis, were generally clustered with the new 5 CIMP markers, MSI and BRAF
mutation. We have provided valuable data for standardization of methylation markers for the detection of CIMP-high in colorectal cancer.
Studying epigenetic and genetic aberrations is important in cancer research 
. We used quantitative PCR assays (MethyLight 
) to determine the degree of DNA methylation, which is robust enough to reproducibly differentiate low-level methylation from high-level methylation 
. Our resource of a large colorectal cancer sample obtained from two large prospective cohorts (representing a relatively unbiased sample compared to a single-hospital-based sample) has provided a sufficient power to evaluate the 16 methylation markers, and to simultaneously assess independent relations of CIMP-high with multiple clinical and tumoral molecular variables.
Interestingly, unsupervised clustering analysis using a large number of tumors revealed that KRAS
mutation was not clustered with any of the 16 methylation markers. However, as shown in our previous studies 
mutation was more common in CIMP-low tumors compared to CIMP-high and CIMP-0 tumors. Although these findings appeared to be discrepant, we believe that KRAS
mutation is perhaps associated with a random pattern of CpG island methylation, indicated by the non-clustering phenomenon in clustering analysis. In contrast, our clustering analysis has clearly shown that BRAF
mutation is clustered with CIMP-high specific markers, indicating that BRAF
mutation is perhaps associated with a non-random pattern of CpG island methylation.
Previous studies identified various factors associated with CIMP-high, including old age, female, proximal location, poor differentiation, mucin, signet ring cells, MSI-high, BRAF
mutation, wild-type KRAS
, inactive β-catenin, wild-type APC
, high LINE-1 methylation level, and wild-type TP53 
. However, many of these factors are interrelated. Thus, in order to properly decipher the relations with CIMP-high, it is necessary to use a large number of tumors, determine a number of molecular features, and perform comprehensive biostatistical analysis. We were able to utilize a large colorectal cancer sample that has been examined for multiple molecular events, and appropriate biostatistical methods. summarizes our current knowledge on the associations of clinical, pathologic and molecular features including CIMP in colorectal cancer. It is very important to keep in mind these relations, when analyzing the association between any of these factors and an outcome (e.g., molecular changes in colorectal cancer, patient mortality, etc.). These factors may confound the relationship of interest. Indeed, we have demonstrated confounding effects of MSI, BRAF
and tumor location in a number of the associations in . In particular, signet ring cells, KRAS
and p53 were no longer associated with CIMP-high after adjusting for the confounders.
We have shown that the relations of CIMP-high with tumor differentiation, KRAS
mutation and LINE-1 hypomethylation appear to differ according to MSI status. MSI is a major molecular classifier in colorectal cancer 
. MSI-high tumors have been shown to exhibit widespread mutations in nucleotide repeat sequences such as those in TGFBR2
and BAX 
. Thus, it is likely that overall genomic changes in MSI-high tumors are dissimilar to those of non-MSI-high tumors. That may explain why there are some pathologic and molecular features that are differentially associated with CIMP-high according to MSI status.
In summary, using the 16 methylation markers and a large population-based sample, we have evaluated performance of each of the 16 methylation markers in an unbiased fashion. Our current study provides valuable data for standardization of the use of CIMP-high-specific markers. Using the validated CIMP-specific methylation marker panel, we have comprehensively analyzed the clinical, pathologic and molecular features of CIMP-high colorectal cancer by comprehensive biostatistical methods. We have provided the rationale to use the validated CIMP-high-specific methylation marker panel in clinical and research settings. Further studies are necessary to elucidate fundamental molecular defects that lead to CIMP-high colorectal cancer.