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Ma et al. Hepatoma Res 2019;5:8 I http://dx.doi.org/10.20517/2394-5079.2018.104 Page 5 of 12

TERT promoter insertional mutations by HBV DNA integration
HBV infection has been shown to be a causative factor of HCC, especially in Asians where chronic hepatitis
B infection is prevalent. Integration of HBV DNA into the human genome of HCC cells is evident in
HBV-related HCC [8,40,48,58-64] . Several lines of evidence demonstrate that the integration sites of HBV are
not random. Integration of certain genomic sites, including near or within the genes of TERT [8,48,59-65] ,
[48]
MLL4 [48,59,61-63,65] and CCNE1 [48,61-63,65] are more frequently identified in HCC .
To date, 13 independent studies have identified a total of 262 integrations of HBV DNA in the TERT
gene, meaning that in more than 20% HBV-related HCC cases, TERT gene is interrupted by HBV
integration [7,58,65-75] . TERT is the most susceptible gene for HBV integration, followed by MLL4 (79
[76]
integrations), CCNE1 (22 integrations) and CCNA2 (19 integrations) . According to our pool analysis of
the results from these articles [7,58,65-75] , among the 262 HBV integrations in TERT, 73.28% (192/262) occur in
the TERT promoter region, including 26% in the core functional fragment (-223 bp to -14 bp from the ATG
translational start site). As the regulation of TERT expression largely depends on the activity of the TERT
promoter region, especially the core functional fragment, HBV integration in the TERT promoter may have
an important functional role in HCC development.

A few studies suggested that HBV tended to integrate in common chromosomal fragile sites, where DNA
replication was delayed and DNA sequences were more susceptible to breakage [63,64] . Nevertheless, the
[64]
findings that TERT was a recurrent integration site but not a fragile site demand new explanation . More
recent studies have therefore presented new possibilities. One study proposed that HBV preferentially
integrates into TERT gene because disruption at these loci lowers the threshold for malignant transformation
[59]
and thus grants a selective advantage to carcinogenesis . Another two studies, using a similar line of
reasoning, suggested that the recurrence of HBV integrations into TERT promoter region in HCC could be
due to the potential growth advantage that augmented TERT expression provides for the clonal expansion
and carcinogenesis of hepatocytes [60,62] . In TCGA database, the HCC with HBV DNA insertion into the
TERT promoter displays the highest level of TERT RNA expression among all HCCs, suggesting an HBV
[48]
cis-activating event did exist .
HBV integrations promote the development of HCC by inducing global genomic instability, elevating
expression of adjacent genes, viral-host fusion transcripts and secondary mutations of host or viral
genes, as well as by DNA copy number variations and proteins with oncogenic activity (X and preS gene
products) [58,61,64,65] . Recently, based on the discovery that both HBV integration and somatic mutations in the
[69]
TERT promoter were more frequent in male patients with HCC, Li et al. proposed a novel mechanism
in which sex hormones, along with GABPA play a role in regulating TERT expression. They analyzed
101 HBV-related HCC cases using a capture-next-generation sequencing platform and concluded with
convincing evidence that the integration of HBV DNA, whose sequence contains both androgen- and
estrogen-responsive elements, into the TERT promoter permits the androgen-receptor to up-regulate and the
[62]
estrogen-receptor to down-regulate TERT transcription in a HNF4α-dependent manner .

OTHER GENOMIC AND EPIGENOMIC ALTERATIONS ON TERT PROMOTER IN HCC
TERT amplification in HCC
[8]
[77]
Totoki et al. showed that TERT focal amplification was detected in 6.7% of the total 608 cases. Schulze et al.
observed less than 5% of TERT focal amplification in the 243 liver tumors. However, while both studies
described the occurrence of TERT focal amplification in HCC, none of them investigated its effect on
TERT expression level. Thus, more research is needed to confirm the role of TERT amplification in liver
carcinogenesis.

Epigenetic modification of TERT promoter in HCC
[78]
As for epigenetic regulation of TERT promoter in HCC, Iliopoulos et al. observed a strong negative
correlation between TERT promoter methylation and TERT expression in all liver tissues they studied,