Comprehensive biostatistical analysis of CpG island methylator phenotype in colorectal cancer using a large population-based sample

Abstract

Background: The CpG island methylator phenotype (CIMP) is a distinct phenotype associated with microsatellite instability (MSI) and BRAF mutation in colon cancer. Recent investigations have selected 5 promoters (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1) as surrogate markers for CIMP-high. However, no study has comprehensively evaluated an expanded set of methylation markers (including these 5 markers) using a large number of tumors, or deciphered the complex clinical and molecular associations with CIMP-high determined by the validated marker panel. METHOLODOLOGY/PRINCIPAL FINDINGS: DNA methylation at 16 CpG islands [the above 5 plus CDKN2A (p16), CHFR, CRABP1, HIC1, IGFBP3, MGMT, MINT1, MINT31, MLH1, p14 (CDKN2A/ARF) and WRN] was quantified in 904 colorectal cancers by real-time PCR (MethyLight). In unsupervised hierarchical clustering analysis, the 5 markers (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1), CDKN2A, CRABP1, MINT31, MLH1, p14 and WRN were generally clustered with each other and with MSI and BRAF mutation. KRAS mutation was not clustered with any methylation marker, suggesting its association with a random methylation pattern in CIMP-low tumors. Utilizing the validated CIMP marker panel (including the 5 markers), multivariate logistic regression demonstrated that CIMP-high was independently associated with older age, proximal location, poor differentiation, MSI-high, BRAF mutation, and inversely with LINE-1 hypomethylation and beta-catenin (CTNNB1) activation. Mucinous feature, signet ring cells, and p53-negativity were associated with CIMP-high in only univariate analysis. In stratified analyses, the relations of CIMP-high with poor differentiation, KRAS mutation and LINE-1 hypomethylation significantly differed according to MSI status.

Conclusions: Our study provides valuable data for standardization of the use of CIMP-high-specific methylation markers. CIMP-high is independently associated with clinical and key molecular features in colorectal cancer. Our data also suggest that KRAS mutation is related with a random CpG island methylation pattern which may lead to CIMP-low tumors.

Figure 1. Hierarchical clustering analysis of 16 methylation makers, MSI, KRAS and BRAF in colorectal cancers.

Horizontal and vertical axes represent markers and cases, respectively. The expanded view of clustering tree for the markers is shown on the right. The 8 markers in our CIMP-high diagnostic panel (CACNA1G, IGF2, RUNX3, MLH1, SOCS1, CDKN2A (p16), CRABP1 and NEUROG1) are clustered closely, supporting that these markers are good CIMP-high markers. Also note the close relationship between MSI, BRAF and the 8 CIMP-high markers. KRAS mutation is not clustered with any of the methylation markers analyzed, suggesting that KRAS mutation (which is associated with CIMP-low , ; see Supplemental Figure) is probably associated with a random methylation pattern.

Figure 2. Smoothing spline for the age/CIMP-high association.

Log_e(OR for CIMP-high) (y axis) according to age (x axis) is shown (with young age as a referent). Broken lines indicate 95% confidence interval. Note the linear relation between age and CIMP-high. CIMP, CpG island methylator phenotype; OR, odds ratio.

Figure 3. Smoothing spline for the LINE-1/CIMP-high association.

Log_e(OR for CIMP-high) (y axis) according to LINE-1 methylation (x axis) is shown (with high-level LINE-1 methylation as a referent). Broken lines indicate 95% confidence interval. Note the inverse linear relation between LINE-1 hypomethylation and CIMP-high. CIMP, CpG island methylator phenotype; LINE-1, long interspersed nucleotide element-1; OR, odds ratio.

Figure 4. Summary of associations of CIMP-high with clinical and molecular features.

The broken line indicates the relatively weak association.