Quantification of circulating HBV RNA expressed from intrahepatic cccDNA in untreated and NUC treated patients with chronic hepatitis B

Abstract

Objective: A convenient, reproducible biomarker of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) transcriptional activity is lacking. We measured circulating HBV RNA (cirB-RNA) in untreated and nucleos(t)ide analogues (NUC) treated chronic hepatitis B (CHB) patients to define its correlation with intrahepatic viral markers and HBV core-related antigen (HBcrAg).

Design: Paired liver biopsy and serum samples were collected from 122 untreated and 30 NUC-treated CHB patients. We measured cirB-RNA, HBV DNA, hepatitis B surface antigen (HBsAg), HBcrAg and alanine aminotransferase levels. cirB-RNA was quantified using an investigational HBV RNA assay for use on the cobas 6800 system. The test detects a region spanning the HBV canonical polyadenylation site. cccDNA and 3.5 kb RNA in liver tissue were assessed by quantitative PCR and droplet digital PCR.

Results: cirB-RNA was detectable in 100% of HBeAg(+) chronic hepatitis (CH), 57% and 14% of HBeAg(-) CH and chronic infection untreated patients and 47% of NUC-treated patients. cirB-RNA undetectability was associated with lower intrahepatic cccDNA transcriptional activity, as well as serum HBcrAg, but no significant differences in HBsAg, in both untreated and treated patients. In untreated HBeAg(-) patients, cirB-RNA correlated with intrahepatic 3.5 kb RNA and cccDNA transcriptional activity, serum HBV DNA and HBcrAg, but not with HBsAg or total cccDNA levels. Combined undetectability of both cirB-RNA and HBcrAg detection in untreated HBeAg(-) patients identified a subgroup with the lowest levels of intrahepatic transcriptionally active cccDNA.

Conclusion: Our results support the usefulness of quantification of circulating HBV RNA expressed from cccDNA as an indicator of intrahepatic active viral reservoir in both untreated and NUC-treated CHB patients.

Trial registration number: NCT02602847.

Keywords: RNA expression; chronic viral hepatitis; hepatitis B; liver biopsy.

Figure 1

cirB-RNA distribution according to disease status (untreated patients). Boxes span the 25th–75th percentile, whiskers span the range and the horizontal bar in the box represents the median. Assay LOD and LLOQ are marked with dotted lines; the shaded area represents undetectability. ****p<0.001. CH, chronic hepatitis; CI, chronic infection; LLOQ, lower limit of quantification; LOD, limit of detection.

Figure 2

Intrahepatic viral markers in patients with detectable versus undetectable cirB-RNA (all untreated patients). Intrahepatic cccDNA (A, B) and 3.5 kb RNA (C, D) results were available for 120 patients. The 3.5 kb RNA/cccDNA ratio (E, F) could be calculated for 113 patients and was different from 0 in 66 patients (49 cirB-RNA(+) and 17 cirB-RNA(−) patients). cccDNA was undetectable in seven patients (six cirB-RNA(+) and one cirB-RNA(−)), while 3.5 kb RNA was undetectable in 54 patients (34 cirB-RNA(+) and 20 cirB-RNA(−)). Assay detection limits are marked with a dotted line; the shaded area represents undetectability. cccDNA, covalently closed circular DNA. ***p<0.001, ****p<0.0001.

Figure 3

Correlations between cirB-RNA and intrahepatic and serum viral markers (HBeAg(−) untreated patients). Intrahepatic cccDNA (A) and 3.5 kb RNA (B) results were available for 88 HBeAg(−) patients. 3.5 kb RNA/cccDNA ratio (C) could be calculated for 82 patients. Only samples having quantifiable values for both intrahepatic HBV markers and cirB-RNA are shown (n=33 for cccDNA, n=29 for 3.5 kb RNA, n=28 for 3.5 kb RNA/cccDNA). cccDNA, covalently closed circular DNA.

Figure 4

cirB-RNA distribution according to HBcrAg levels (untreated patients). Boxes span the 25th–75th percentile, whiskers span the range, and the horizontal bar in the box represents the median. Assay LOD and LLOQ are marked with dotted lines; the shaded area represents undetectability. HBcrAg, HBV core-related antigen; HBV, hepatitis B virus; LLOQ, lower limit of quantification; LOD, limit of detection.

Figure 5

Integrative analysis of serum HBcrAg and cirB-RNA reveals subsets of HBeAg(−) patients with discordant quantification. Principal component analysis (PCA) score plot, where each dot represents a patient. Colours distinguish distribution of HBcrAg(−) cirB-RNA(−) (blue dots), HBcrAg(−) cirB-RNA(+) (fuchsia dots), HBcrAg(+) cirB-RNA(−) (orange dots) and HBcrAg(+) cirB-RNA(+) (grey dots). Distribution of cccDNA (B, C) and 3.5 kb RNA (D, E) levels according to the patients’ groups derived from the combination of HBcrAg and cirB-RNA positivity and showed in (A). cccDNA, covalently closed circular DNA; HBcrAg, HBV core-related antigen; HBV, hepatitis B virus; LOD, limit of detection.

Figure 6

cirB-RNA and intrahepatic viral markers in NUC-treated patients. Intrahepatic cccDNA (A, B), 3.5 kb RNA (C, D) and 3.5 kb RNA/cccDNA ratio (E, F) in NUC-treated patients with undetectable (neg) or detectable (pos) cirB-RNA. Boxes span the 25th–75th percentile, whiskers span the range, and the horizontal bar in the box represents the median. Assay detection limits are marked with dotted lines; the shaded area represents undetectability. cccDNA, covalently closed circular DNA; LOD, limit of detection; NUC, nucleos(t)ide analogues.