Cell‐Free Virus‐Host Chimera DNA From Hepatitis B Virus Integration Sites as a Circulating Biomarker of Hepatocellular Cancer

Abstract

BaCKgRoUND aND aIMS: Early recurrence of hepatocellular carcinoma (HCC) after surgical resection compromises patient survival. Timely detection of HCC recurrence and its clonality is required to implement salvage therapies appropriately. This study examined the feasibility of virus-host chimera DNA (vh-DNA), generated from junctions of hepatitis B virus (HBV) integration in the HCC chromosome, as a circulating biomarker for this clinical setting. appRoaCH aND ReSUltS: HBV integration in 50 patients with HBV-related HCC was determined by the Hybridization capture-based next-generation sequencing (NGS) platform. For individual HCC, the vh-DNA was quantified by specific droplet digital PCR (ddPCR) assay in plasma samples collected before and 2 months after surgery. HBV integrations were identified in 44 out of 50 patients with HBV-related HCC. Tumor-specific ddPCR was developed to measure the corresponding vh-DNA copy number in baseline plasma from each patient immediately before surgery. vh-DNA was detected in 43 patients (97.7%), and the levels correlated with the tumor sizes (detection limit at 1.5 cm). Among the plasma collected at 2 months after surgery, 10 cases (23.3%) still contained the same signature vh-DNA detected at baseline, indicating the presence of residual tumor cells. Nine of them (90%) experienced HCC recurrence within 1 year, supporting vh-DNA as an independent risk factor in predicting early recurrence. Analysis of circulating vh-DNA at recurrence further helped identify the clonal origin. A total of 81.8% of recurrences came from original HCC clones sharing the same plasma vh-DNA, whereas 18.2% were from de novo HCC. CoNClUSIoNS: vh-DNA was shown to be a circulating biomarker for detecting the tumor load in majority of patients with HBV-related HCC and aided in monitoring residual tumor and recurrence clonality after tumor resection. (Hepatology 2020;72:2063-2076). S urgical resection has been recommended for earlystage hepatocellular carcinoma (HCC) when the liver function is well preserved. (1) Although the clinical outcome is improved after resection, these patients still confront high risk for recurrent HCC, at a rate of about 40%-50% within 2 years. (2) Recurrence after resection, especially those that recur within 1 year, significantly decreases the survival of patients with HCC. Early recurrence after tumor resection, possibly originating from residual HCC, thus compromises the long-term prognosis.

Droplet digital PCR of viral ‎DNA/RNA, current progress, challenges, and future perspectives

“…147 (C) PCR-based CRISPR-Cas13a system. 171 (D, E) A highly efficient microfluidic droplet-based platform to screen single virus particles for optimum vaccine candidate antigenic compounds. Like the standard ELISA method, HIV-1 particles were incubated with alkaline phosphatase (AP)-labeled broadly neutralizing antibody PGT128.…”

“…CD4+ T cells were washed and mixed 1:1 with ddPCR master mix and cell lysis agent. Droplets generated, and cells are lysed inside the droplets, followed by PCR amplification of Tat‐Rev spliced or unspliced cell‐associated HIV‐1 RNA 170 . The schematic diagram shows a rapid, targeted, and culture‐free viral infectivity assay in drop‐based microfluidics.…”

Unique Features of Hepatitis B Virus-Related Hepatocellular Carcinoma in Pathogenesis and Clinical Significance

“…A novel ctDNA biomarker released from HBV-related HCC tumors was recently proposed to overcome these limitations, namely, the circulating DNA fragment generating from the junctions of HBV integration in the chromosomes of HCC. This virus–host chimera DNA (vh–DNA) consists of the junctional fragments at the HBV integration site, containing both virus and human DNA sequences [ 149 ]. Since the randomly distributed HBV DNA integrations have been identified in ~90% of HBV-related HCC [ 19 ], the integration-derived vh–DNA can be applied as a biomarker for detecting the majority of HBV–HCC.…”

“…At the same time, their detection levels correlated with the tumor sizes (with detection limit at 1.5 cm diameter). By monitoring the residual circulating vh–DNA in the blood collected after surgery, 90% of the patients with detectable vh–DNA levels in 2 months postsurgery experienced HCC recurrence within 1 year [ 149 ]. Therefore, the vh–DNA generated by HBV integration might become a new circulating ctDNA biomarker for detecting the tumor load in most HBV-related HCC patients.…”

Viral Biomarkers for Hepatitis B Virus-Related Hepatocellular Carcinoma Occurrence and Recurrence

“…A study of 20 people with HBV-HCC found circulating vh-DNA representing 87 different HBV integration sites, which were enriched in genes involved in cancer-related pathways, suggesting they could act as a biomarker for HCC diagnosis (Li et al, 2019). Moreover, Li et al (2020) detected vh-DNA in 97.7% of people with HBV-related HCC. Two months following HCC resection, the same vh-DNA sequence could be detected in 10 cases (23.3%), nine of whom (90%) experienced HCC recurrence within a year.…”