The DNA hypermethylation phenotype of colorectal cancer liver metastases resembles that of the primary colorectal cancers

BMC Cancer. 2020 Apr 6;20(1):290. doi: 10.1186/s12885-020-06777-6.


Background: Identifying molecular differences between primary and metastatic colorectal cancers-now possible with the aid of omics technologies-can improve our understanding of the biological mechanisms of cancer progression and facilitate the discovery of novel treatments for late-stage cancer. We compared the DNA methylomes of primary colorectal cancers (CRCs) and CRC metastases to the liver. Laser microdissection was used to obtain epithelial tissue (10 to 25 × 106 μm2) from sections of fresh-frozen samples of primary CRCs (n = 6), CRC liver metastases (n = 12), and normal colon mucosa (n = 3). DNA extracted from tissues was enriched for methylated sequences with a methylCpG binding domain (MBD) polypeptide-based protocol and subjected to deep sequencing. The performance of this protocol was compared with that of targeted enrichment for bisulfite sequencing used in a previous study of ours.

Results: MBD enrichment captured a total of 322,551 genomic regions (249.5 Mb or ~ 7.8% of the human genome), which included over seven million CpG sites. A few of these regions were differentially methylated at an expected false discovery rate (FDR) of 5% in neoplastic tissues (primaries: 0.67%, i.e., 2155 regions containing 279,441 CpG sites; liver metastases: 1%, i.e., 3223 regions containing 312,723 CpG sites) as compared with normal mucosa samples. Most of the differentially methylated regions (DMRs; 94% in primaries; 70% in metastases) were hypermethylated, and almost 80% of these (1882 of 2396) were present in both lesion types. At 5% FDR, no DMRs were detected in liver metastases vs. primary CRC. However, short regions of low-magnitude hypomethylation were frequent in metastases but rare in primaries. Hypermethylated DMRs were far more abundant in sequences classified as intragenic, gene-regulatory, or CpG shelves-shores-island segments, whereas hypomethylated DMRs were equally represented in extragenic (mainly, open-sea) and intragenic (mainly, gene bodies) sequences of the genome. Compared with targeted enrichment, MBD capture provided a better picture of the extension of CRC-associated DNA hypermethylation but was less powerful for identifying hypomethylation.

Conclusions: Our findings demonstrate that the hypermethylation phenotype in CRC liver metastases remains similar to that of the primary tumor, whereas CRC-associated DNA hypomethylation probably undergoes further progression after the cancer cells have migrated to the liver.

Keywords: Colorectal cancer; CpG islands; CpG sites; DNA methylation; Differentially methylated regions; Liver metastasis; MBD capture; Methyl-binding domain; Normal colorectal mucosa.

MeSH terms

  • Biomarkers, Tumor / genetics*
  • Colorectal Neoplasms / genetics*
  • Colorectal Neoplasms / metabolism
  • Colorectal Neoplasms / pathology*
  • Epigenesis, Genetic
  • Epigenome*
  • High-Throughput Nucleotide Sequencing / methods
  • Humans
  • Laser Capture Microdissection / methods
  • Liver Neoplasms / genetics
  • Liver Neoplasms / metabolism
  • Liver Neoplasms / secondary*
  • Phenotype
  • Promoter Regions, Genetic


  • Biomarkers, Tumor