The Landscape of Cell-Free HBV Integrations and Mutations in Cirrhosis and Hepatocellular Carcinoma Patients

Abstract

Purpose: Intratumoral hepatitis B virus (HBV) integrations and mutations are related to hepatocellular carcinoma (HCC) progression. Circulating cell-free DNA (cfDNA) has shown itself as a powerful noninvasive biomarker for cancer. However, the HBV integration and mutation landscape on cfDNA remains unclear. Experimental Design: A cSMART (Circulating Single-Molecule Amplification and Resequencing Technology)-based method (SIM) was developed to simultaneously investigate HBV integration and mutation landscapes on cfDNA with HBV-specific primers covering the whole HBV genome. Patients with HCC (n = 481) and liver cirrhosis (LC; n = 517) were recruited in the study. Results: A total of 6,861 integration breakpoints including TERT and KMT2B were discovered in HCC cfDNA, more than in LC. The concentration of circulating tumor DNA (ctDNA) was positively correlated with the detection rate of these integration hotspots and total HBV integration events in cfDNA. To track the origin of HBV integrations in cfDNA, whole-genome sequencing (WGS) was performed on their paired tumor tissues. The paired comparison of WGS data from tumor tissues and SIM data from cfDNA confirmed most recurrent integration events in cfDNA originated from tumor tissue. The mutational landscape across the whole HBV genome was first generated for both HBV genotype C and B. A region from nt1100 to nt1500 containing multiple HCC risk mutation sites (OR > 1) was identified as a potential HCC-related mutational hot zone. Conclusions: Our study provides an in-depth delineation of HBV integration/mutation landscapes at cfDNA level and did a comparative analysis with their paired tissues. These findings shed light on the possibilities of noninvasive detection of virus insertion/mutation.

ViMIC: a database of human disease-related virus mutations, integration sites and cis-effects

“…Recently, the study of Zheng et al. has used paired samples to draw the landscape of VISs and VMs in HBV-associated cirrhosis and hepatocellular carcinoma ( 28 ). Our work will contribute to and help researchers who are interested in gene regulation mechanisms to systematically investigate the functional annotation of VISs in the host genome.…”

“…An understanding of viral pathogenic mutations and extraction of feature patterns mined from virus sequence data will improve effective surveillance and early detection for high-risk virus-infected diseases ( 25 , 26 ). Significant viral mutations/regions for affecting integration reactions ( 10 , 11 , 13 , 27 ) and the comparative analysis of virus integration/mutation landscapes at cfDNA level and paired tissues ( 28 ) have been reported. Previous studies have also demonstrated that transcriptional regulators contribute to the control of viral replication and regulate critical cis-elements in a virus ( 29 , 30 ).…”

Liquid Biopsy Using Cell-Free or Circulating Tumor DNA in the Management of Hepatocellular Carcinoma

“… 48 Notably, the number of HBV integration events in cfDNA of HCC patients is correlated with ctDNA concentration. 49 The plasma level of virus–host chimera DNA generated by HBV integration into the human genome is associated positively with tumor size. Therefore, HBV integration detected in cfDNA could be a circulating biomarker for prognostic prediction before tumor resection as well as monitoring residual or recurrent tumor after resection.…”

“… 58 For HBV integration, most of the mismatch breakpoints were regarded as random and sporadic events, except TERT and KMT2B breakpoints, which showed high concordance between HCC tissue and plasma cfDNA. 49 …”

“…Simultaneous detection of HBV integration landscape and mutations in cfDNA by circulating single-molecule amplification and resequencing technology–based targeted deep sequencing showed a higher AUC value when combined with AFP than that with AFP alone, indicating superior detection or diagnostic power for HCC. 49 Plasma cfDNA CNV discovered by shallow whole-genome sequencing, when combined with fragment size and AFP, facilitates early detection of HCC by achieving 92.6% accuracy and better performance than other predictors. 32 …”