DNA methylation profiles of primary colorectal carcinoma and matched liver metastasis

Abstract

Background: The contribution of DNA methylation to the metastatic process in colorectal cancers (CRCs) is unclear.

Methods: We evaluated the methylation status of 13 genes (MINT1, MINT2, MINT31, MLH1, p16, p14, TIMP3, CDH1, CDH13, THBS1, MGMT, HPP1 and ERα) by bisulfite-pyrosequencing in 79 CRCs comprising 36 CRCs without liver metastasis and 43 CRCs with liver metastasis, including 16 paired primary CRCs and liver metastasis. We also performed methylated CpG island amplification microarrays (MCAM) in three paired primary and metastatic cancers.

Results: Methylation of p14, TIMP3 and HPP1 in primary CRCs progressively decreased from absence to presence of liver metastasis (13.1% vs. 4.3%; 14.8% vs. 3.7%; 43.9% vs. 35.8%, respectively) (P<.05). When paired primary and metastatic tumors were compared, only MGMT methylation was significantly higher in metastatic cancers (27.4% vs. 13.4%, P = .013), and this difference was due to an increase in methylation density rather than frequency in the majority of cases. MCAM showed an average 7.4% increase in DNA methylated genes in the metastatic samples. The numbers of differentially hypermethylated genes in the liver metastases increased with increasing time between resection of the primary and resection of the liver metastasis. Bisulfite-pyrosequencing validation in 12 paired samples showed that most of these increases were not conserved, and could be explained by differences in methylation density rather than frequency.

Conclusions: Most DNA methylation differences between primary CRCs and matched liver metastasis are due to random variation and an increase in DNA methylation density rather than de-novo inactivation and silencing. Thus, DNA methylation changes occur for the most part before progression to liver metastasis.

Figure 1. DNA methylation status of thirteen cancer-specific or age-related genes/CpG islands in primary CRCs without and with liver metastasis.

Each dot represents the methylation level of an individual sample. Horizontal lines represent mean methylation levels for each group. *, P = .0005; **, P = .0113; #, P = .0452. LM-, primary CRCs without liver metastasis; LM+, primary CRCs with liver metastasis. CRCs, colorectal cancers.

Figure 2

A) DNA methylation status of thirteen cancer-specific or age-related genes/CpG islands in 16 primary CRCs and matched liver metastasis. Each dot represents the methylation level of an individual sample. Horizontal lines represent mean methylation levels for each group. ¶, P = .013. Primary, primary CRCs; Mets, liver metastasis. B) DNA methylation and mutation status in 16 primary CRCs and paired liver metastases. Each column represents a separate gene locus indicated on top. Each row represents a primary or metastatic tumor. Average methylation density of less than 15% are shown in green, 15 to 50% in yellow and over 50% in red. Black square, presence of mutation; white square, absence of mutation; ND, not detected; NA, not applicable. C) Differences in methylation between primary CRCs and matched liver metastases. Red boxes denote an increase in methylation levels at metastatic tumors of more than two times higher when compared with primary tumors and the methylation level of at least one of the tumors is greater than 15%; green boxes show decrease of methylation levels at metastatic tumors of more than two times lower than primary tumors and the methylation level of at least one of the tumors is greater than 15%. White boxes are all others.

Figure 3. Microarray analysis of hypermethylated genes in liver metastatic cancers.

A) The Venn diagram shows the overlap and differences in methylated genes of liver metastasis in three patients. A total number of 6528 genes were analyzed by 18340 microarray probes recognizing promoter CpG islands. B) Dendrogram and heat map overview of unsupervised hierarchical cluster analysis of DNA methylation in liver metastatic cancers of three patients. Each cell represents DNA methylation status of a gene in an individual sample. Red and green in cells reflect high and low methylation level, respectively, as shown in the scale bar (log₂-transformed scale) below the matrix.

Figure 4. DNA methylation analysis for eight genes identified by MCAM in 12 paired primary CRCs and liver metastasis.

Each column represents a separate gene locus indicated on top. Each row represents a primary or metastatic tumor, normal tissue type or colon cancer cell. Average methylation densities of less than 15% are shown in white, 15 to 50% in gray and over 50% in black. PBL, peripheral blood.