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Liver Cancer Marked by Both Noncoding, Structural Mutations, Study Finds

NEW YORK (GenomeWeb) – A team of Japanese researchers has characterized noncoding and structural variants present in liver cancer.

The development of liver cancer has been linked to a number of factors including hepatitis B and hepatitis C viral infections, alcohol use, metabolic disease, and carcinogen exposure. This, the researchers noted, suggests that there may be genomic heterogeneity in the disease.

The Riken Center for Integrative Medical Science's Hidewaki Nakagawa and his colleagues performed whole-genome and RNA sequencing on some 300 liver cancer and normal tissue samples from Japanese patients. As they reported in Nature Genetics today, they uncovered mutational signatures linked to carcinogenesis and recurrent mutations in both coding and noncoding regions. In addition, the researchers noted a number of cancer-related genes affected by structural variants within the liver tumor samples.

"These comprehensive and integrative analyses show that [structural variants] and noncoding mutations have biological relevance and affect gene expression and regulatory functions," the authors wrote in their paper.

The samples the team analyzed were made up of mostly hepatocellular carcinomas, but also some intra-hepatic cholangiocarcinomas and a handful of combined cHCC/ICC types. They also included cases from an HCV-positive hepatitis background, an HBV-positive hepatitis background, and from a combined HBV-positive and HCV-positive hepatitis background, as well as cases negative for both viruses.

They sequenced the tumor samples to an average 39.2-fold depth to uncover an average 9,718 point mutations, 271 indels, 40.6 structural variants, and 2.5 HBV viral integration sites.

From this, Nakagawa and his colleagues uncovered seven mutational signatures from within their set of hepatocellular carcinoma samples. While most of these signatures were highly similar to those listed in the Catalog of Somatic Mutations in Cancer, one signature dubbed W6 — characterized by T to A mutations — appeared to be a new signature.

At the same time, the researchers also detected a number of viral integration sites within the patients' tumors. Seventy-four samples had more than 10 reads that mapped to the HBV genome, and they uncovered 223 HBV integration events, with recurrent integration sites near the TERT, KMT2B, and SOX5 genes. In addition to reads originating from HBV, they also uncovered ones from HCV, adenovirus, and some bacterial genomes.

The researchers also reported a number of recurrent mutations. Some 25 genes — including TP53, ARID2, RB1, and HNF4A — had a significant number of protein-altering mutations, and 14 of the 25 were enriched for loss-of-function mutations, indicating genes that help suppress tumors.

A number of noncoding elements like the TERT promoter were also enriched for mutations, according to the researchers. TERT promoter mutations, they added, were more common in hepatocellular carcinomas than in intra-hepatic cholangiocarcinomas.

Through a slightly different approach, the researchers also uncovered more than 100 regions with significant local mutations, including exons of the known cancer genes TP53, CTNNB1, and NFE2L2. They also noted regions upstream of the coding regions of TERT, WDR74, and MED16 had significant mutations.

They further homed in on four consecutive regions on chromosome 18 with recurrent mutations. These recurrent mutations were present in 25 samples, as well as in 10 percent of samples from another liver cancer cohort and in two HCC cell lines. However, Nakagawa and his colleagues noted, the consequences of this mutation are unclear.

Structural variations also appear to play a role in liver cancer, the researchers said. They found that tandem duplications were more common among early replicating regions, while deletions were more common among late replicating regions, consistent with its accessibility to DNA-repair factors.

Structural variants commonly affected genes like TERT, CDKN2A, and TTC2 and could influence the expression of nearby genes, including MET, APC, and PTPN11, the authors wrote, adding that both structural variations and HBV integration in TERT and its promoter region were linked to higher gene expression levels.

When they folded together their data on point mutations, indels, structural variations and copy number alterations, the researchers found 38 genes that harbored a significantly large number of mutations. While some of these were known driver genes like CCND1 and CDKN2A or were chromatin regulators like ASH1L or NCOR1, eight were new candidate genes. Knockdown experiments of these in liver cancer cell lines indicated that seven of them affected cell growth.

A number of these mutations could be tied to clinical factors, Nakagawa and his colleagues said. For instance, TERT promoter mutations were linked with smoking, while CTTNB1 mutations were associated with HCV and LRP1B mutations with HCV and alcohol consumption.

These mutations also formed clusters. Cluster one cases, marked by ARID 2 and PBRM1 mutations, and cluster six cases, marked by TP53 mutations, exhibited worse prognosis, while cluster 3, with MACROD2 mutation, showed better prognosis.