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Researchers Use WGS, RNA-Seq to Characterize Colorectal Cancer Mutational Landscape

NEW YORK (GenomeWeb) – An international team of researchers has used whole-genome sequencing, RNA sequencing, and methylation arrays to investigate the intra-tumor mutational landscape of colorectal cancer at the single-cell level.

In a paper published today in Nature, researchers from the UK, the Netherlands, and the US described their efforts to characterize organoids derived from multiple single cells from three colorectal cancers as well as from adjacent normal intestinal crypts. "Colorectal cancer cells showed extensive mutational diversification and carried several times more somatic mutations than normal colorectal cells. Most mutations were acquired during the final dominant clonal expansion of the cancer and resulted from mutational processes that are absent from normal colorectal cells," the authors wrote.

They also found that intra-tumor diversification of DNA methylation and transcriptome states occurred, and that the alternations were cell-autonomous, stable, and followed the phylogenetic tree of each cancer. Further, when the researchers exposed the tumors to anti-cancer drugs, they observed that even closely related cells in the same tumor had markedly different responses, indicating that colorectal cancer cells experience substantial increases in the rates of their somatic mutations as compared to normal colorectal cells. "Genetic diversification of each cancer is accompanied by pervasive, stable, and inherited differences in the biological states of individual cancer cells," the team added.

The researchers dissected colorectal cancers from three previously untreated individuals and derived organoid cultures from them. They then sequenced the coding regions of 360 known cancer genes in all normal and cancer-derived clonal organoids for likely driver mutations, and a subset were whole-genome sequenced. Somatic mutations were identified by comparison with the sequences of DNA extracted from pieces of normal colorectal tissue. The researchers also analyzed the clonal organoids for DNA methylation at 470,000 CpG sites, performed RNA sequencing, and assessed them for response to several anti-cancer therapeutics.

They found a mean of 3,792 base substitutions in normal organoid clones derived from the first patient, 3,172 from the second patient, and 3,621 from the third patient. The mean number of base substitutions in cancer-derived clones was higher in all three individuals: 72,398 in patient one, 22,291 in patient two, and 14,209 in patient three.

There were also substantial differences in the number of small indels and genome rearrangements — a mean of 227 small indels in clones derived from normal colorectal epithelium from patient one, 130 from patient two, and 167 from patient three. By comparison, the mean number of indels was 27,893 in cancer clones from patient one, 1,485 from patient two, and 2,021 from patient three. There was a mean of one genome rearrangement in clonal organoids derived from normal colorectal epithelial cells compared with means in cancer-derived clonal organoids of 71 rearrangements from patient one, 176 from patient two, and 67 from patient three.

The researchers also extracted mutational signatures from each patient-derived cell line and found eight base substitution mutational signatures, including seven that have been previously described and one that was novel.

"Each mutational signature can be regarded as the outcome of a mutational process, which includes components of DNA damage or modification, DNA repair (or absence of it), and DNA replication, with each component potentially influencing the profile of the signature," the authors wrote. "The numbers of mutations of several signatures differed markedly between individual branches [of the phylogenetic tree], indicating varying contributions of mutational processes in different parts of the cancer."

The team's methylation array experiments, meanwhile, showed that the methylation states of normal colorectal stem cells from the different individuals were relatively similar, but tumors from different individuals had developed divergent epigenetic states. Further, their RNA-seq analysis found that diversification of methylation and transcriptome state occurred within each cancer and this was apparently heritable, stable, and independent of the tumor microenvironment, as it persisted after the organoids were cultured in vitro.

"To our knowledge, this is the first systematic and integrated analysis at genetic, epigenetic, transcriptomic, and functional levels of multiple single-cell-derived clones from human cancers to incorporate high-quality and comprehensive description of essentially all somatic mutations present in multiple single cells," the authors concluded. "Future studies analyzing the genomes, methylomes, and transcriptomes of primary cells of a cancer will be needed to reveal all genetic, epigenetic, and transcriptional variation occurring between cancer cells in vivo. Nevertheless, this study has shown the strength of the organoid system in stably retaining these characteristics and enabling functional assays on clones derived from individual cells."

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