The use of a high-sensitivity droplet digital PCR assay for risk prediction in chronic hepatitis B

Abstract

Hepatitis B virus (HBV) DNA is essential for guiding the management of chronic hepatitis B (CHB). Droplet digital PCR (ddPCR) is a sensitive method that may improve HBV DNA detection over conventional assays. This thesis aimed to develop a high-sensitivity ddPCR assay for serum HBV DNA, and to utilize the assay for risk prediction in CHB.

A ddPCR serum HBV DNA assay with lower limit of detection and quantification of 1.6 IU/ml and 9.4 IU/ml respectively was developed. The assay had excellent specificity (96.2%), linearity (R2=0.988), intra-run variability (mean coefficient of variation 0.69%), and inter-run variability (mean coefficient of variation 4.54%).

The ddPCR assay was utilized in four clinical studies. The first study recruited 208 patients on nucleos(t)ide analogues (NUCs) (104 HCC patients matched with 104 non-HCC controls), and with serum HBV DNA suppressed to unquantifiable levels by conventional assays. Detectable serum HBV DNA by the high-sensitivity ddPCR assay (residual viremia) was identified in 65.9% of patients. Residual viremia within 2 years of HCC diagnosis independently predicted HCC development (OR 4.965, 95%CI 2.626-9.389, p<0.001), and this association remained robust in sensitivity analysis at different timepoints.

The second study included 63 patients with HBsAg seroclearance (21 HCC patients matched with 42 non-HCC controls). Residual viremia was detected in 57.1% of patients, and was independently associated with HCC development (OR 11.894, 95% CI 1.604-88.197, p=0.015).

The third study focused on 104 NUC-treated HBV-related HCC patients. Residual viremia within 2 years independently predicted more advanced HCC staging on presentation (OR 0.292 for presenting with very early-stage HCC, 95% CI 0.097-0.881, p=0.029). Residual viremia was also associated with microvascular invasion (44.4% vs 9.1% in patients without residual viremia, p=0.033), a histological indicator of HCC aggressiveness. Residual viremia independently predicted liver-related mortality after median follow-up for 7.2 years (HR 4.723, 95% CI 1.267-17.599, p=0.021).

The fourth study included 63 NUC-treated CHB patients without HCC. These patients had vibration controlled transient elastography with liver steatosis and fibrosis determined by controlled attenuation parameter (CAP) and liver stiffness respectively. Residual viremia within 2 years was inversely associated with hepatic steatosis (OR 0.183, 95% CI 0.062-0.544, p=0.002). Increasing incidences of residual viremia within 2 years was associated with lower CAP values (Median CAP 268 dB/m, 233 dB/m, 226 dB/m and 226 dB/m in patients with no residual viremia, residual viremia at 1 timepoint, 2 timepoints and 3 timepoints respectively, p=0.009 for trend). Residual viremia had no significant associations with liver fibrosis.

To conclude, ddPCR is a powerful tool for HBV DNA detection, particularly when conventional HBV DNA assays have limited detection value (i.e. in patients on NUCs or with HBsAg seroclearance). In NUC-treated patients, residual viremia was associated with HCC development, more aggressive HCC, and poorer liver-related mortality after HCC diagnosis. Residual viremia also had an inverse association with steatosis. In patients with HBsAg seroclearance, residual viremia was an independent predictor of HCC. Our findings established the role of residual viremia for risk stratification. The application of ddPCR in HBV will be beneficial towards our goal of hepatitis elimination.