Three DNA methylation epigenotypes in human colorectal cancer.
OBJECTIVE
Whereas the CpG island methylator phenotype (CIMP) in colorectal cancer associates with microsatellite instability (MSI)-high and BRAF-mutation(+), the existence of an intermediate-methylation subgroup associated with KRAS-mutation(+) is controversial, and suitable markers for the subgroup have yet to be developed. Our aim is to clarify DNA methylation epigenotypes of colorectal cancer more comprehensively.


METHODS
To select new methylation markers on a genome-wide scale, we did methylated DNA immunoprecipitation-on-chip analysis of colorectal cancer cell lines and re-expression array analysis by 5-aza-2'-deoxycytidine/Trichostatin A treatment. Methylation levels were analyzed quantitatively in 149 colorectal cancer samples using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Colorectal cancer was epigenotyped by unsupervised two-way hierarchical clustering method.


RESULTS
Among 1,311 candidate silencing genes, 44 new markers were selected and underwent quantitative methylation analysis in colorectal cancer samples together with 16 previously reported markers. Colorectal cancer was clustered into high-, intermediate-, and low-methylation epigenotypes. Methylation markers were clustered into two major groups: group 1 showing methylation in high-methylation epigenotype, and group 2 showing methylation in high- and intermediate-methylation epigenotypes. A two-step marker panel deciding epigenotypes was developed with 95% accuracy: the 1st panel consisting of three group-1 markers (CACNA1G, LOX, SLC30A10) to extract high-methylation epigenotype, and the 2nd panel consisting of four group-2 markers (ELMO1, FBN2, THBD, HAND1) and SLC30A10 again to divide the remains into intermediate- and low-methylation epigenotypes. The high-methylation epigenotype correlated significantly with MSI-high and BRAF-mutation(+) in concordance with reported CIMP. Intermediate-epigenotype significantly correlated with KRAS-mutation(+). KRAS-mutation(+) colorectal cancer with intermediate-methylation epigenotype showed significantly worse prognosis.


CONCLUSIONS
Three methylation epigenotypes exist in colorectal cancer, and suitable classification markers have been developed. Intermediate-methylation epigenotype with KRAS-mutation(+) correlated with worse prognosis.
Yagi
Koichi
K
Genome Science Division, Department of Gastrointestinal Surgery, Research Center for Advanced Science and Technology, Translational Systems Biology and Medicine Initiative, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Akagi
Kiwamu
K
Hayashi
Hiroshi
H
Nagae
Genta
G
Tsuji
Shingo
S
Isagawa
Takayuki
T
Midorikawa
Yutaka
Y
Nishimura
Yoji
Y
Sakamoto
Hirohiko
H
Seto
Yasuyuki
Y
Aburatani
Hiroyuki
H
Kaneda
Atsushi
A
D014408:Biomarkers, Tumor / Q000032:analysis; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics*; D018899:CpG Islands; D019175:DNA Methylation; D011905:Genes, ras; D005838:Genotype; D006801:Humans; D010641:Phenotype
D014408:Biomarkers, Tumor