# Quantification of Circulating HBV RNA Expressed from Intrahepatic cccDNA in Untreated and NUC Treated Patients with Chronic Hepatitis B

Barbara Testoni, Caroline Scholtès, Marie-Laure Plissonnier, Alexia Paturel, Françoise Berby, Floriana Fachetti, François Villeret, Elisabetta Degasperi, Beth Scott, Aaron Hamilton, et al.

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Barbara Testoni, Caroline Scholtès, Marie-Laure Plissonnier, Alexia Paturel, Françoise Berby, et al. Quantification of Circulating HBV RNA Expressed from Intrahepatic cccDNA in Untreated and NUC Treated Patients with Chronic Hepatitis B. Gut, 2024, 73 (4), pp.659-667. 10.1136/gutjnl-2023-330644. hal-04598137

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Original research

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

Barbara Testoni,¹,² Caroline Scholtès,¹,²,³ Marie-Laure Plissonnier,¹ Alexia Paturel,¹,²

Françoise Berby,¹ Floriana Facchetti,⁴ François Villeret,¹,²,⁵ Elisabetta Degasperi,⁴

Beth Scott,⁶ Aaron Hamilton,⁶ Marinha Heil,⁶ Pietro Lampertico,¹,⁴,⁷

Massimo Levrero,¹,²,⁵,⁸ Fabien Zoulim,¹,²,⁵

► Additional supplemental material is published online only. To view, please visit the journal online (http://dx.doi.org/ 10.1136/gutjnl-2023-330644).

For numbered affiliations see end of article.

**Correspondence to**
Professor Fabien Zoulim, Cancer Research Centre Lyon, Lyon 69008, France;
fabien.zoulim@inserm.fr
Dr Barbara Testoni;
barbara.testoni@inserm.fr

Presented at The International Liver Congress 2021 (Poster P-408).

Received 6 July 2023
Accepted 26 September 2023
Published Online First
25 October 2023

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► http://dx.doi.org/10.1136/gutjnl-2023-331217

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© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

**To cite:** Testoni B, Scholtès C, Plissonnier M-L, et al. Gut 2024;73:659–667.

## 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.

## INTRODUCTION

It is estimated that 296 million people were infected with hepatitis B virus (HBV) globally in 2015.¹ HBV causes chronic hepatitis (CH) and is

## WHAT IS ALREADY KNOWN ON THIS TOPIC

⇒ Measurement of serum hepatitis B virus (HBV) RNA concentration has been proposed has a promising surrogate marker for intrahepatic HBV replication.

⇒ The lack of a commercially available, standardised and automated assay hampers the implementation of serum HBV RNA quantification in clinical practice.

## WHAT THIS STUDY ADDS

⇒ Serum HBV RNA levels quantified by using a new automated prototype assay significantly correlated with intrahepatic covalently closed circular DNA (cccDNA) transcriptional activity in both untreated and nucleos(t)ide analogues-treated chronic hepatitis B patients.

⇒ The prototype assay, whose target region includes the canonical HBV polyadenylation site, preferentially detects RNA derived from cccDNA vs transcripts originating from integrated sequences.

⇒ The distribution of serum HBV RNA and HBV core-related antigens detection shows an incomplete overlap in HBeAg(−) patients.

## HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY

⇒ Measurement of serum HBV RNA levels with the automated prototype assay might represent a new non-invasive, robust and reproducible diagnostic tool to guide treatment cessation and monitor intrahepatic viral clearance.

associated with serious liver disease and cancer. In infected hepatocytes the virus replicates through a covalently closed circular DNA (cccDNA) intermediate, which serves as the template for transcription of viral RNA for protein production and generation of new viral genomes through reverse transcription.² HBV-infected patients are managed clinically according to disease stage based on detection and measurement of several host and viral biomarkers in serum including alanine aminotransferase (ALT) levels, the degree of liver inflammation, hepatitis
B surface antigen (HBsAg) and viral DNA.<sup>3 4</sup> Antiviral therapy is based on one or more inhibitors of the viral reverse transcriptase (nucleos(t)ide analogues, NUCs). Response to antiviral therapy is monitored through measurement of the different biomarkers.<sup>3 4</sup> However, complete clearance of HBV cannot be achieved with current treatments because of the persistence of residual cccDNA in hepatocytes. This can lead to resurgence of viraemia in patients who stop antiviral therapy prematurely. Alternative treatment strategies aimed at HBV cure<sup>5</sup> must eliminate the cccDNA reservoir or silence it, the measurement of which is challenging since it requires invasive sampling methods. A diagnostic tool that provides information to guide treatment cessation and monitor intrahepatic viral clearance and that is non-invasive (eg, uses convenient specimen types such as peripheral blood), standardised, amenable to automation, robust and reproducible, is needed. The advantages and disadvantages of several candidate biomarkers have been reviewed elsewhere.<sup>6 7</sup> Briefly, HBeAg and serum HBV DNA concentrations lose their correlation with intrahepatic cccDNA levels after HBeAg seroconversion or NUC treatment, respectively. The fact that HBsAg can be derived from viral sequences integrated in the host genome also undermines its value in predicting cccDNA levels and transcriptional activity, particularly in HBeAg(−) patients.<sup>8 9</sup>

Circulating HBV RNA (cirB-RNA) concentration is a relatively
novel biomarker that may be useful for antiviral treatment moni-
toring. Pregenomic RNA (pgRNA) concentration in blood has been
proposed to reflect transcription from cccDNA in infected hepato-
cytes.<sup>10–13</sup> Serum HBV RNA concentrations, measured mainly with
research laboratory developed assays, have good predictive power
for both on-treatment serological response and off-treatment dura-
bility.<sup>14–18</sup> The combination of undetectable cirB-RNA and HBV
core-related antigen (HBcrAg) at the end of treatment is more predic-
tive for sustained suppression of replication off-treatment compared
with either biomarker alone.<sup>14</sup>

Here, we measured cirB-RNA levels by using a new automated,
prototype quantitative HBV RNA assay in both untreated and NUC-
treated patients to assess the relationship between cirB-RNA concen-
tration and other viral markers in blood and in hepatocytes that are
associated with cccDNA transcriptional activity.

METHODS

Patients and sampling

Serum samples and core liver biopsies were collected at the same time in 122 untreated chronic hepatitis B (CHB) patients enrolled at the Hepatology Unit of the ‘Hospices Civils de Lyon’ and at the Gastroenterology and Hepatology Division of the ‘Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico’ in Milan. Part of the liver tissue was snap frozen and stored at –80°C. Patients were serologically negative for hepatitis C virus, HIV and hepatitis D virus.

Thirty NUC-treated patients who were enrolled in the ECOGR-EFFE French prospective cohort¹⁹ and underwent liver transplantation were also enrolled in this study. All received NUC therapy (90% with entecavir or tenofovir) for a median of 2.6 years. Part of the explanted liver collected from an hepatocellular carcinoma (HCC)-free region surrounding the lesion was snaped frozen, stored at -80°C and available for further virologic investigations.

Patient and public involvement

Patients were not involved in the design and conduct of this
research, since not appropriate to the retrospective nature of the
study.

**Laboratory assays for viral biomarkers in serum and liver**

cirB-RNA concentrations in serum were quantified by real-time PCR using an investigational HBV RNA assay for use on the cobas 6800/8800 Systems (cobas HBV RNA, Roche Diagnostics, Pleasanton, California, USA). cobas HBV RNA is a sensitive nucleic acid test for detection and quantification of HBV RNA in EDTA plasma or serum, with limit of detection (LOD) and lower limit of quantification (LLOQ) of 5 and 10 copies/mL, respectively. The difference between DNA and RNA concentrations in untreated patients ranged from 1.5 to 3.4 log10 copies/mL.²⁰ LOD was confirmed in clinical samples representing HBV genotypes A, B, C, D, E. Assay specificity for RNA versus DNA is enhanced with a blocking oligo that binds to the HBV DNA genome in competition with an assay primer. The assay is calibrated in units of copies/mL, based on a synthetic armoured (arRNA) Roche internal standard spanning 435 bp derived from the 3' end of HBV RNA and quantitated by droplet digital PCR (ddPCR). The dynamic range of quantification using arRNA was 10⁻¹⁰ copies/mL. Results from clinical samples were linear between 10 and 10⁷ RNA copies/mL for all genotypes tested. Detectable but not quantifiable results (above the LOD but below the LLOQ) and undetectable results were assigned arbitrary values of 5 and 1 copies/mL, respectively, for illustrative purposes. This prototype assay amplifies HBV transcripts containing the canonical polyadenylation signal²¹²² and thus preferentially quantifies RNA derived from cccDNA. All tests were performed by trained operators in accordance with the manufacturers' specifications using the cobas 6800 system.

Serum HBV DNA viral load was quantified using either the Roche (Roche Diagnostics, Mannheim, Germany) or Abbott quantitative assay (Abbott Diagnostic, Chicago, USA). The LOD for serum HBV DNA was 10 IU/mL for the Roche assay and 20 IU/mL for the Abbott assay. HBsAg quantification was carried out by conventional methods using either Roche (Roche Diagnostics, Mannheim, Germany) or Abbott Architect assay (Abbott Diagnostic, Chicago, USA) with a LLOQ set at 0.05 IU/mL.

Serum HBcrAg concentrations were measured in serum using the Lumipulse G HBcrAg assay on the Lumipulse G600II Analyzer (Fujirebio Europe, Gent, Belgium) according to the manufacturer's instructions (LOD 2.8 log₁₀ U/mL).<sup>23</sup> Samples for which the HBcrAg concentration was≥7.0 log<sub>10</sub> U/mL were diluted with a manufacturer-supplied dilution reagent and retested. Undetectable results were assigned arbitrary values of 2.0 U/mL for illustrative purposes.

cccDNA and 3.5 kb RNA concentrations in liver biopsy samples were assessed by quantitative PCR and ddPCR as previously described.<sup>24 25</sup> 3.5 kb RNA primers and probes recognise both pgRNA and pre-Core RNA, thus we collectively refer the identified transcripts as ‘3.5 kb RNA’. cccDNA transcriptional activity was estimated as the 3.5 kb RNA/cccDNA ratio. Beta-globin DNA and β-glucuronidase (GUSB) RNA quantification served as internal references for cccDNA copies/cell and 3.5 kb RNA relative quantity expression, respectively. The cccDNA assay LOD is 3×10<sup>-6</sup> copies/cell. Undetectable cccDNA and 3.5 kb RNA results were assigned arbitrary values of 3×10<sup>-6</sup> copies/cell and 10<sup>-4</sup> units, respectively, for illustrative purposes.

**Histological analysis**

Fibrosis and necroinflammatory activity were quantified using the METAVIR classification<sup>26</sup> by the pathology services of Hospices Civils de Lyon and ‘Foundation IRCCS Ca’ Granda Ospedale Maggiore Policlinico’ in Milan. Patients were divided into two groups for both necroinflammatory activity and fibrosis scores:
none or mild (below or equal to 1) versus moderate to severe (above 2), according to the European Association for the Study of the Liver (EASL) practice guidelines for HBV treatment.³

## HBV integration harbouring hepatoma cell culture

Hep3B cells were cultured in Dulbecco's modified Eagle's medium (DMEM, ThermoFischer Scientific, Waltham, Massachusetts, USA) supplemented with L-Glutamine (ThermoFischer Scientific) sodium pyruvate (ThermoFischer Scientific), 5% fetal calf serum (Fetalclone II, ThermoFischer Scientific), 100 U/mL penicillin (ThermoFischer Scientific) and 100 µg/mL streptomycin (ThermoFischer Scientific) at 37°C and 5% CO₂. For PLC/PRF/5 cells, Eagle's Minimum Essential Medium (ATCC) was used as a base medium with 10% heat-inactivated FBS (ThermoFischer Scientific). RNAs from cellular extracts of both cell lines were extracted using the Roche manual workflow²⁰ and quantified by the cobas HBV RNA assay and by an in-house ddPCR assay (see online supplemental material).

## Statistical analysis

All data were analysed using Prism V.9.3.0 (GraphPad Software, San Diego, USA). Statistical correlations were tested using the

Pearson test for normal variables and the Spearman test for other variables. Categorical variables were compared using the χ² or Fischer's exact tests and quantitative variables were compared using the Student's t-test or non-parametric tests (Mann-Whitney or Kruskall-Wallis), as appropriate. Principal component analysis (PCA) was conducted on all viral replication markers in the serum (HBV DNA, qHBsAg and HBcrAg concentrations) and in the liver (HBV-DNA, cccDNA, 3.5 kb RNA concentrations and cccDNA transcriptional activity), as well as on parameters of liver injury (ALT concentration, fibrosis and necroinflammatory activity score). PCA was computed on the first five dimensions using Qlucore Omics Explorer software (Qlucore, Sweden).

## RESULTS

### Untreated CHB patients

The untreated cohort was composed of 122 patients, 32 HBeAg(+) and 90 HBeAg(−), and was predominantly male; the median age was 40 years (table 1). Six HBV genotypes were represented, with the majority (55.5%) belonging to genotype A or D. Patients had mild levels of fibrosis and necroinflammatory activity (35 to 44% had scores of 2 or more). While all 32 HBeAg(+) patients had detectable cirB-RNA, we defined two

<table><caption>**Table 1** Untreated patient characteristics</caption><thead><tr><th>Variable</th><th></th><th>Total cohort (n=122)</th><th>HBeAg(+) cirB RNA(+) (n=32)</th><th>HBeAg(−) cirB RNA(+) (n=51)</th><th>HBeAg(−) cirB RNA(−) (n=39)</th><th>P value*</th></tr></thead><tbody><tr><td rowspan="2">Age†</td><td>Years</td><td>40.2<br>(28.6-50.1)</td><td>32.4<br>(24-43.8)</td><td>44.2<br>(30.4-52.5)</td><td>43.3<br>(33.8-54.4)</td><td>ns‡</td></tr><tr><td>M/F (% M)</td><td>86/36 (70%)</td><td>25/7 (78%)</td><td>36/15 (70%)</td><td>25/14 (64%)</td><td>ns§</td></tr><tr><td rowspan="5">Origin</td><td>Europe</td><td>42 (34%)</td><td>12 (38%)</td><td>18 (35%)</td><td>12 (31%)</td><td rowspan="5">–</td></tr><tr><td>Asia</td><td>26 (21%)</td><td>10 (32%)</td><td>9 (18%)</td><td>7 (18%)</td></tr><tr><td>Sub-Saharan Africa</td><td>25 (20%)</td><td>3 (9%)</td><td>11 (21%)</td><td>11 (28%)</td></tr><tr><td>North Africa</td><td>16 (13%)</td><td>4 (13%)</td><td>7 (14%)</td><td>5 (13%)</td></tr><tr><td>Middle East</td><td>13 (11%)</td><td>3 (9%)</td><td>6 (12%)</td><td>4 (10%)</td></tr><tr><td rowspan="7">Viral genotype</td><td>A</td><td>20 (16%)</td><td>9 (28%)</td><td>5 (10%)</td><td>6 (15.4%)</td><td rowspan="7">ns§</td></tr><tr><td>B</td><td>3 (2%)</td><td>1 (3%)</td><td>0 (0%)</td><td>2 (5%)</td></tr><tr><td>C</td><td>11 (9%)</td><td>7 (23%)</td><td>3 (6%)</td><td>1 (3%)</td></tr><tr><td>D</td><td>48 (39%)</td><td>9 (28%)</td><td>29 (57%)</td><td>10 (26%)</td></tr><tr><td>E</td><td>14 (11%)</td><td>1 (3%)</td><td>6 (12%)</td><td>7 (18%)</td></tr><tr><td>F</td><td>3 (2%)</td><td>2 (6%)</td><td>0 (0%)</td><td>1 (3%)</td></tr><tr><td>ND</td><td>23 (9%)</td><td>3 (9%)</td><td>8 (15%)</td><td>12 (31%)</td></tr><tr><td rowspan="2">Viral load†</td><td>log₁₀ IU/mL</td><td>4.4 (2.9-7.2)</td><td>8.0 (7.4-8.6)</td><td>4.5 (3.5-6.1)</td><td>3.0 (2.4-5.3)</td><td>&lt;0.0001‡</td></tr><tr><td>U/L</td><td>52.5 (34-79.2)</td><td>96.5 (67-203.3)</td><td>50 (35.5-65.5)</td><td>39 (26.5-65.0)</td><td>0.007‡</td></tr><tr><td>HBsAg†</td><td>log₁₀ IU/mL</td><td>3.9 (3.4-4.3)</td><td>4.6 (3.9-5.2)</td><td>3.8 (3.1-4.0)</td><td>4.1(3.4-4.5)</td><td>ns‡</td></tr><tr><td rowspan="2">HBcrAg**</td><td>N detectable (%)</td><td>90 (75%)</td><td>31 (97%)</td><td>41 (80%)</td><td>19 (49%)</td><td>0.001§</td></tr><tr><td>log₁₀ U/mL†,**</td><td>4.8 (3.8-7.5)</td><td>8 (7.3-8.4)</td><td>4.1 (3.7-5.2)</td><td>3.5 (3.1-4)</td><td>0.004‡</td></tr><tr><td>cirB RNA†</td><td>log₁₀ copies/mL</td><td>3.9 (2-5.4)</td><td>5.4 (4.8-6.1)</td><td>2.3 (1.5-3.7)</td><td>–</td><td>–</td></tr><tr><td>Fibrosis score</td><td>N (%) with score ≥2</td><td>55 (44%)</td><td>17 (53%)</td><td>28 (55%)</td><td>10 (26%)</td><td>0.009§</td></tr><tr><td>Activity score</td><td>N (%) with score ≥2</td><td>43 (35%)</td><td>17 (53%)</td><td>19 (37%)</td><td>7 (18%)</td><td>ns‡</td></tr><tr><td rowspan="3">CHB phase††</td><td>HBeAg(+) CH</td><td>32</td><td>32</td><td>0</td><td>0</td><td rowspan="3">&lt;0.0001‡</td></tr><tr><td>HBeAg(−) CI</td><td>29</td><td>0</td><td>6</td><td>23</td></tr><tr><td>HBeAg(−) CH</td><td>61</td><td>0</td><td>45</td><td>16</td></tr></tbody></table>

*Comparing cirB RNA(+) versus cirB RNA(−) in HBeAg(−) patients.

†Data are expressed as median (1st–3rd quartile) of valid results.

‡Mann-Whitney U test, α threshold=0.5.

§Fischer exact test or χ² test.

¶Data available for 119 patients.

**Data available for 120 patients.

††According to the European Association for the Study of the Liver (EASL) 2017 guidelines.

–, insufficient data to calculate; CH, chronic hepatitis; CHB, chronic hepatitis B; CI, chronic infection; HBcrAg, HBV core-related antigen; ns, not significant.
<figure><img src="image_5.png" /><figcaption>**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.</figcaption></figure>

groups of HBeAg(−) patients based on detection of cirB-RNA (table 1). HBeAg(−) patients with detectable cirB-RNA (n=51) had higher serum HBV DNA, HBcrAg, and ALT levels, percentage with detectable HBcrAg, and fibrosis score of 2 or more, compared with HBeAg(−) patients with undetectable cirB-RNA (n=39; table 1).

### cirB-RNA concentrations and CHB disease phases

Detectability and concentrations of cirB-RNA were assessed in patients classified as having CH or chronic infection (CI).³ All HBeAg(+) CH patients had quantifiable (>LLOQ) cirB-RNA, compared with only 57% of HBeAg(−) CH and 14% of HBeAg(−) CI patients (figure 1). The median of quantifiable cirB-RNA concentrations was much higher in CH HBeAg(+) patients compared with CH or CI HBeAg(−) patients. In the subgroup of six CI HBeAg(−) patients with quantifiable cirB-RNA, levels of serum HBV DNA, HBsAg, HBcrAg and intrahepatic cccDNA were similar to those with undetectable cirB-RNA (Mann-Whitney U test, p values >0.1; data not shown).

The pattern of differences in serum HBV DNA and HBcrAg concentrations between the three groups was similar to that observed for cirB-RNA (online supplemental figure 1). However, HBsAg concentrations in HBeAg(−) CI patients were about 10-fold lower than HBeAg(+) CH patients and were similar to HBeAg(−) CH patients (online supplemental figure 1). cirB-RNA levels were higher in patients with high versus low ALT levels, necroinflammatory activity or fibrosis scores (online supplemental figure 2). No differences were observed according to HBV genotype (online supplemental figure 3).

### cirB-RNA and intrahepatic viral markers

Intrahepatic markers of viral replication were compared in patients with detectable versus undetectable cirB-RNA. Patients with undetectable cirB-RNA had lower levels of cccDNA (median 0.06 vs 0.56 copies/cell), 3.5 kb RNA (median 0.03 vs 21.9) and cccDNA transcriptional activity (3.5 kb RNA/ccDNA ratio 1.1 vs 17.7), compared with patients with detectable cirB-RNA (figure 2). Similar results were obtained in the HBeAg(−) subset of patients (online supplemental figure 4). HBcrAg and HBV DNA concentrations in serum were significantly higher in HBeAg(−) patients with detectable versus undetectable

<figure><img src="image_6.png" /><figcaption>**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/ccDNA 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.</figcaption></figure>

cirB-RNA, while no significant difference was found in HBsAg levels (see table 1).

In HBeAg(−) patients, cirB-RNA concentrations were positively correlated with levels of intrahepatic 3.5 kb RNA (r=0.77, p<0.001), cccDNA transcriptional activity (r=0.78, p<0.0001), serum HBV DNA (r=0.81, p<0.0001) and serum HBcrAg (r=0.73, p<0.0001), but not with intrahepatic cccDNA or serum HBsAg levels (figure 3), contrary to HBeAg(+) group, where cirB-RNA was significantly correlated with both cccDNA and serum HBsAg levels (online supplemental figure 5), as previously observed with in-house assays.²⁷ ²⁸ In the whole cohort, cirB-RNA concentration positively and significantly correlated with all viral intrahepatic and serum markers (online supplemental figure 6).
<figure><img src="image_7.png" /><figcaption>**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.</figcaption></figure>

In HBeAg(−) patients, the predominant source of HBsAg is HBV DNA integrated in the host cell genome.⁸ ⁹ Therefore, the lack of correlation between cirB-RNA and serum HBsAg suggests that the cobas HBV RNA assay preferentially detects viral RNAs produced by cccDNA. This is supported by in vitro data showing that the assay does not detect intracellular HBV RNA in cell lines with integrated HBV DNA but devoid of cccDNA (PLC/PRF/5 and Hep3B cells; online supplemental figure 7).

## Two groups of HBeAg(−) CH patients defined by cirB RNA levels

In CH HBeAg(−) patients, there appeared to be a grouping of patients with cirB-RNA above or below approximately 3 log₁₀ copies/mL (eg, **figure 3**). Intrahepatic 3.5 kb RNA levels, 3.5 kb RNA/cccDNA ratio, liver fibrosis scores and necro-inflammatory activity were significantly higher in the patients with cirB-RNA concentration over 3 log₁₀ copies/mL, while cccDNA quantity and HBsAg levels were not different (online supplemental figure 8).

### cirB-RNA distribution according to HBcrAg levels

cirB-RNA and HBcrAg levels in serum have both been proposed as markers of intrahepatic viral replication or transcription, and are correlated with each other. As shown in **figure 4**, median cirB-RNA levels and the proportion with detectable cirB-RNA both increased in groups of patients with increasing HBcrAg levels, irrespective of their HBeAg status. To further investigate the relationship between HBcrAg, cirB-RNA and the viral and clinical parameters, we performed PCA in HBeAg(−) patients. Four profiles are recognisable in the PCA scatterplot in **figure 5A**, where each point represents a sample from one patient. The HBcrAg(+) cirB-RNA(+) group (grey dots) is clearly spatially differentiated from the other three groups. This can be explained by the higher serum HBV DNA levels in these samples (**table 2**). Nine out of 30 (30%) patients with undetectable HBcrAg had detectable cirB-RNA (fuchsia dots). This subgroup of patients was mainly composed of HBeAg(−) CH patients (7 out of 9) and had increased intrahepatic cccDNA levels (median 0.16 vs 0.04 copies/cell, p=0.04) compared with the 21 patients with undetectable cirB-RNA and HBcrAg (blue dots in panel A) (**figure 5B,C**). 3.5 kb RNA levels were also higher, but the difference was not statistically significant

<figure><img src="image_8.png" /><figcaption>**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.</figcaption></figure>

(median 0.05 vs 0.03, p=0.1) (**figure 5E**). Conversely, 17 of 39 (44%) cirB-RNA(−) patients had detectable HBcrAg (orange dots in panel A). Most of them (11 out of 17) were HBeAg(−) CI patients, and showed higher cccDNA amount (0.10 vs 0.04 copies/cell, p=0.02), compared with those with undetectable cirB-RNA and HBcrAg (**figure 5B,C**). Also, median of 3.5 kb RNA (median 0.13 vs 0.03, p=0.5) was higher, but did not reach statistical significance (**figure 5E**).

## NUC-treated patients

Characteristics of the 25 NUC-treated patients at the time of liver transplantation are summarised in **table 3**. At the time of sampling, 18 of the 25 NUC-treated patients had hepatocellular carcinoma. Treatment with entecavir, tenofovir or lamivudine for a median of 29 months preceded liver transplant and tissue sampling (**table 3**).

All treated patients had detectable HBsAg, 19 of 25 (76%) had detectable HBcrAg and 11 of 25 (44%) had detectable cirB-RNA (**table 3**). No baseline clinical characteristics were associated with cirB-RNA positivity. While only three (12%) treated patients had detectable HBV DNA in serum, most had detectable cccDNA (19 of 25, 76%) and 3.5 kb RNA (17 of 25, 68%) in their liver biopsy sample (**table 3**). Quantities of intrahepatic HBV DNA and RNA were low (less than 0.05 copies per cell), consistent with other data from patients receiving long term NUC therapy.²⁴

Compared with the cirB-RNA(+) NUC-treated patients, the 14 cirB-RNA(−) patients had similar cccDNA levels but lower 3.5 kb RNA levels (median 0.001 vs 0.04, p=0.04; **figure 6C,D**). The 3.5 kb RNA/ccDNA ratio was also lower (median 0.16 vs 15.8), although the difference was not statistically significant, possibly due to the small sample size. Two of five patients negative for both cirB-RNA and HBcrAg had detectable intrahepatic cccDNA and 3.5 kb RNA by ddPCR).

HBsAg (median 4.9 vs 6.1 log₁₀ IU/mL, p=0.002) and HBcrAg levels (3.8 vs 4.9 log₁₀ U/mL, p=0.005) were also higher in cirB-RNA(+) patients, but cirB-RNA levels were not correlated with serum HBsAg (R=0.3, p=ns) (data not shown).
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

**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.

DISCUSSION

Emerging treatment strategies aimed at HBV cure must elim-
inate or, at least, permanently inactivate the intrahepatic
cccDNA reservoir.²⁹ The size of the cccDNA pool, as well as
its capacity to produce HBV RNA, varies across CHB phases
and during NUC treatment.⁹ ²⁴ ³⁰ Therefore, accurate assessment
of the intrahepatic HBV reservoir must include quantification
of cccDNA levels and transcriptional activity. Evaluation of this
reservoir requires sampling of the liver, which is hindered by the

invasiveness of the procedure and the potential for sampling bias
and suboptimal storage procedures.³¹ Measurement of serum
biomarkers that reflect cccDNA activity based on its transcrip-
tional output, the viral RNAs, would represent an invaluable
diagnostic tool to guide treatment cessation and monitor intrahe-
patic viral clearance, especially if it was standardised, automated,
robust and reproducible. Here, using matched liver biopsies and
serum samples, we demonstrate that results from an automated,
prototype quantitative assay that detects preferentially HBV
RNAs expressed from cccDNA²⁰ are able to reflect the intrahe-
patic pool of transcriptionally active cccDNA in untreated and
NUC-treated CHB patients.

Our results indicate that cirB-RNA concentrations in serum
were significantly correlated with other markers of viral replica-
tion in the serum and in the liver (figures 2 and 3). Moreover, in
HBeAg(−) patients, where the transcriptional activity of cccDNA
is decreased and, potentially, not all cccDNA molecules in the
liver are transcriptionally active,⁹ ³⁰ cirB-RNA concentration was
highly correlated with intrahepatic 3.5 Kb HBV RNA levels and
cccDNA transcriptional activity. Interestingly, in this category of
patients, cirB-RNA concentration did not correlate with serum
HBsAg levels. Since HBsAg production is mainly ascribed to inte-
grated HBV DNA rather than cccDNA in HBeAg(−) patients,⁸ ⁹
this results suggest that the cobas HBV RNA assay preferentially
recognises transcripts derived from the cccDNA template. This
assumption is consistent with the design of the assay, since its
target region includes the canonical HBV polyadenylation site, a
region which is lost during integration of viral sequences in the
host genome. This is supported by the observation that the assay
does not detect HBV RNA produced from cell lines containing
only integrated HBV DNA in vitro (online supplemental figure
7). At this stage, we cannot exclude that the cobas assay would
not detect cccDNA-derived transcript that would be truncated
upstream of the canonical polyA region.³² ³³

HBcrAg is another biomarker in serum that reflects intra-
hepatic cccDNA transcriptional activity.<sup>34-36</sup> In our cohort of
untreated patients, cirB-RNA and HBcrAg concentrations were
strongly correlated with each other, in both HBeAg(−) and
HBeAg(+) patients. However, PCA helped to highlight subsets
of patients having discordant detection of the two biomarkers.
Integrative analysis with intrahepatic viral markers demon-
strated that patients with detectable HBcrAg or cirB-RNA had
higher liver cccDNA and HBV RNA levels compared with those
scoring negative for both (table 2). Whether the discrepancy in
the results of the two biomarkers is related to technical differ-
ences in the assays or to a difference in underlying biological or
clinical phenomena remains to be investigated.

Importantly, our study included analysis of difficult to obtain
liver samples from NUC-treated patients. We had access to
serum and liver samples from patients who were eligible for liver
transplantation,¹⁹ and showed correlations between circulating
HBV RNA and cccDNA transcriptional activity. Since serum
HBV RNA concentration is correlated with transcriptionally
active cccDNA in the liver, this surrogate biomarker could play
an important role in evaluation of the efficacy of emerging treat-
ment strategies in new proof-of-concept clinical trials, since most
of them will be conducted in NUC-suppressed patients. Similar
to what we observed for HBcrAg,³⁴ we identified a positive,
and most likely, indirect correlation between cirB-RNA levels
and markers of liver damage. Indeed, higher cirB-RNA levels to
more severe scores of fibrosis and necro-inflammatory activity
and to ALT levels >2N (online supplemental figure 2). It is note-
worthy that cirB-RNA levels correlate with cccDNA transcrip-
tional activity even in patients with HBV replication suppressed
<table><caption>**Table 2** Serum and intrahepatic viral markers of HBeAg(−) patients according to combined HBcrAg and cirB-RNA analysis</caption><thead><tr><th></th><th>HBcrAg(+) cirB-RNA(+) (n=42)</th><th>HBcrAg(+) cirB-RNA(−) (n=17)</th><th>HBcrAg(−) cirB-RNA(+) (n=9)</th><th>HBcrAg(−) cirB-RNA(−) (n=22)</th></tr></thead><tbody><tr><td>Serum HBV DNA (log<sub>10</sub> IU/mL)</td><td>4.85 (3.91–6.28)</td><td>2.96 (2.35–3.77)</td><td>3.21 (2.93–4.48)</td><td>2.69 (2.21–3.03)</td></tr><tr><td>Serum HBsAg (log<sub>10</sub> IU/mL)</td><td>3.86 (3.28–4.09)</td><td>4.05 (3.15–4.49)</td><td>3.5 (2.90–3.77)</td><td>3.64 (3.15–4.15)</td></tr><tr><td>cccDNA positive/total (copies/cell)</td><td>37/42<br>0.13 (0.07–0.31)</td><td>15/17<br>0.10 (0.02–0.62)</td><td>8/9<br>0.16 (0.04–0.84)</td><td>22/22<br>0.04 (0.02–0.11)</td></tr><tr><td>3.5 kb RNA positive/total (units)</td><td>26/42<br>2.46 (0.47–85.7)</td><td>7/17<br>0.13 (0.03–0.15)</td><td>5/9<br>0.05 (0.02–0.09)</td><td>8/22<br>0.03 (0.02–0.04)</td></tr></tbody><tfoot><tr><td colspan="5">cccDNA, covalently closed circular DNA; HBcrAg, HBV core-related antigens; HBV, hepatitis B virus.</td></tr></tfoot></table>

by NUCs and with severe liver damage undergoing OLT for HCC and/or end-stage liver disease.

Our study has several limitations. Relatively small sample sizes for some analyses may have limited our ability to discern

small differences in biomarker concentrations between groups of patients. Given the cross-sectional nature of this study, further investigation is needed to confirm the contribution of the

<table><caption>**Table 3** NUC-treated patient characteristics</caption><thead><tr><th>Variable</th><th></th><th>Total cohort (n=25)</th><th>cirB-RNA(+) (n=11)</th><th>cirB-RNA(−) (n=14)</th><th>P value*</th></tr></thead><tbody><tr><td>Age at LT†</td><td>Years</td><td>57.4 (47.5–64.2)</td><td>58.7 (50.5–64.3)</td><td>56.5 (46.2–64.6)</td><td>ns</td></tr><tr><td>Sex</td><td>M/F (%M)</td><td>21/4 (84%)</td><td>8/3 (73%)</td><td>13/1 (93%)</td><td></td></tr><tr><td rowspan="5">Origin</td><td>Sub-Saharan Africa</td><td>9 (36%)</td><td>4 (36%)</td><td>5 (36%)</td><td rowspan="5">–</td></tr><tr><td>Europe</td><td>9 (36%)</td><td>6 (55%)</td><td>3 (21%)</td></tr><tr><td>Asia</td><td>4 (16%)</td><td>0</td><td>4 (29%)</td></tr><tr><td>North Africa</td><td>1 (4%)</td><td>0</td><td>1 (7%)</td></tr><tr><td>Other</td><td>2 (8%)</td><td>1 (9%))</td><td>1 (7%)</td></tr><tr><td>BMI†</td><td>kg/m<sup>2</sup></td><td>24 (22–26.8)</td><td>26.5 (25.4–38.7)</td><td>23 (21.2–24)</td><td>ns</td></tr><tr><td>Time between HBV diagnosis and LT†</td><td>Months</td><td>6.4 (2.8–12.3)</td><td>5.4 (2.5–10.2)</td><td>6.7 (3.2–15.9)</td><td>ns</td></tr><tr><td>MELD at LT registration†</td><td></td><td>10.6 (6–20.9)</td><td>10.6 (6–20)</td><td>10.5 (6–21.8)</td><td>ns</td></tr><tr><td rowspan="3">CHILD-PUGH score at LT registration</td><td>Class A</td><td>10 (40%)</td><td>4 (36%)</td><td>6 (42%)</td><td rowspan="3">ns</td></tr><tr><td>Class B</td><td>8 (32%)</td><td>4 (36%)</td><td>4 (29%)</td></tr><tr><td>Class C</td><td>7 (28%)</td><td>3 (28%)</td><td>4 (29%)</td></tr><tr><td>Hepatocellular carcinoma (HCC)</td><td>No (%)</td><td>18 (72%)</td><td>9 (82%)</td><td>9 (64%)</td><td>ns</td></tr><tr><td>Active HCC at LT</td><td>No (%)</td><td>3/18 (17%)</td><td>2/9 (22%)</td><td>1/9 (11%)</td><td>ns</td></tr><tr><td>Serum AFP levels1</td><td></td><td>5 (3–15)</td><td>5.5 (3–18.6)</td><td>4 (2.5–13.5)</td><td>ns</td></tr><tr><td rowspan="4">HBV treatment before LT</td><td>Duration (months)†</td><td>29 (11–65)</td><td>29 (15–33)</td><td>34.5 (10.3–72.5)</td><td>ns</td></tr><tr><td>Entecavir</td><td>10 (40%)</td><td>5 (45%)</td><td>5 (36%)</td><td>ns</td></tr><tr><td>Tenofovir</td><td>14 (56%)</td><td>6 (55%)</td><td>8 (57%)</td><td>ns</td></tr><tr><td>Lamivudine</td><td>1 (4%)</td><td>0</td><td>1 (7%)</td><td>ns</td></tr><tr><td rowspan="2">Virological status at LT</td><td>HBsAg</td><td>N detectable (%)<br>log<sub>10</sub> IU/mL†</td><td>25 (100%)<br>5.8 (4.7–6.1)</td><td>11 (100%)<br>6.1 (5.8–6.3)</td><td>14 (100%)<br>4.9 (4.2–5.8)</td><td>0.002</td></tr><tr><td>HBcAg</td><td>N detectable (%)<br>log<sub>10</sub> IU/mL†</td><td>2 (7%)<br>19 (76%)<br>4.2 (3.7–5.1)</td><td>1 (9%)<br>10 (91%)<br>4.9 (4.3–5.3)</td><td>1 (7%)<br>9 (64%)<br>3.8 (3.5–4.1)</td><td>–</td></tr><tr><td rowspan="2">Serum HBV DNA</td><td>N detectable (%)<br>log<sub>10</sub> IU/mL†</td><td>3 (12%)<br>3 (2.3–3.7)</td><td>2 (18%)</td><td>1 (7%)</td><td rowspan="2">–</td></tr><tr><td>cirB-RNA</td><td>N detectable (%)<br>log<sub>10</sub> copies/mL†</td><td>11 (44%)<br>1.6 (1.2–2.6)</td><td>11 (100%)<br>1.6 (1.2–2.6)</td><td>0</td></tr><tr><td>Intrahepatic cccDNA</td><td>N detectable (%)<br>copies/cell†</td><td>19 (76%)<br>0.002 (0.0009–0.009)</td><td>9 (82%)<br>0.002 (0.001–0.02)</td><td>10 (71%)<br>0.002 (0.0007–0.007)</td><td>ns</td></tr><tr><td>Intrahepatic 3.5 kb RNA</td><td>N detectable (%)<br>units†</td><td>17 (68%)<br>0.008 (0.002–0.06)</td><td>8 (73%)<br>0.04 (0.02–68.4)</td><td>9 (64%)<br>(0.001–0.006)</td><td>0.04</td></tr><tr><td>Intrahepatic 3.5 kb RNA/ccDNA</td><td>N detectable (%)<br>units†</td><td>16 (64%)<br>4 (0.5–48.3)</td><td>8 (73%)<br>27.6 (4.2–68.4)</td><td>8 (57%)<br>1.2 (0.35–4.7)</td><td>ns</td></tr></tbody></table>

<footnote>*Comparing cirB RNA(+) versus cirB RNA(−).</footnote>

<footnote>†Data are expressed as median (1st–3rd quartile) of valid results.</footnote>

<footnote>–, insufficient data to calculate; BMI, body mass index; cccDNA, covalently closed circular DNA; HBcrAg, HBV core-related antigens; HBV, hepatitis B virus; LT, liver transplantation; MELD, model for end-stage liver disease score; ns, not significant; NUC, nucleos(t)ide analogues.</footnote>
<figure><img src="image_14.png" /><figcaption>**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.</figcaption></figure>

proposed prototype quantitative HBV RNA assay in the context
of patient follow-up and prediction of functional cure.

In conclusion, our results represent the first study to correlate
intrahepatic viral markers with serum HBV RNA levels, measured
with an automated and standardised assay in untreated and
NUC-treated CHB patients. These data suggest that measure-
ment of serum HBV RNA represents a non-invasive diagnostic
tool to monitor the liver viral reservoir.

Author affiliations

**Correction notice** This article has been corrected since it published Online First.
The labelling in figures 2, 5 and 6 have been corrected.

Twitter Barbara Testoni @TestoniResearch

**Acknowledgements** Manuscript preparation assistance was provided by Data First Consulting (Sebastopol, CA) and funded by Roche. COBAS is a trademark of Roche. All other product names and trademarks are the property of their respective owners.

**Contributors** Conceptualisation: BT, ML and FZ; Data curation: BT, CS, M-LP and FB; Formal analysis: BT, BS and AH; funding acquisition: ML, MH and FZ; investigation: BT, CS, M-LP, AP and FB; resources: FF, FV, ED, MH, PL, ML and FZ; supervision: BT, ML and FZ; visualisation: BT, ML and FZ; writing—original draft: BT, ML and FZ; writing—review and editing: all authors; guarantors: BT and FZ.

**Funding** This work is supported by public grants overseen by the French National Research Agency (ANR) as part of the second 'Investissements d'Avenir' program (reference: ANR-17-RHUS-0003) and by the European Union (grant EU H2020-847939-IP-cure-B) to FZ and ML.

**Competing interests** BS, AH and MH are employees and stockholders of Roche Molecular Systems (Pleasanton, CA, USA). ED served as speaker for Gilead and Abbvie, participated in advisory board of Gilead, Abbvie and Roche, received grant from Gilead and travel grant from Gilead and Advanz Pharma. PL served as speaker and/or participated in advisory board for Roche Pharma/Diagnostics, Gilead Sciences, GSK, Abbvie, Janssen, Myr, Eiger, Antios, Aligos, Vir, Grifols, Altona, Roboscreen. FZ received consulting fees from: Aligos, Antios, Assembly, Gilead, GSK; FZ and BT received research funding to INSERM from: Assembly, Beam Therapeutics, Blue Jay and Janssen. FZ is an Associate Editor of the journal.

**Patient and public involvement** Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

**Patient consent for publication** Not applicable.

**Ethics approval** This study involves human participants and all untreated patients provided written informed consent and the protocol was approved by the Lyon (CPP Sud Est IV 11/040, 30 May 2011, ref Avis A11-168) and Milan (ref. CirB RNA, 25 May 2020) institutional Ethic Committees. The study of NUC-treated patients was conducted in accordance with the relevant ethical standards and approved by the local advisory committee (IRB: CPP Sud-Est IV (ref: A-12-158); ID RCB: 2012-A00383-40). Participants gave informed consent to participate in the study before taking part.

**Provenance and peer review** Not commissioned; externally peer reviewed.

**Data availability statement** All data relevant to the study are included in the article or uploaded as online supplemental information.

**Supplemental material** This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.

**Open access** This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

ORCID iDs

Barbara Testoni http://orcid.org/0000-0001-5588-5465
François Villeret http://orcid.org/0000-0002-9327-4590
Pietro Lampertico http://orcid.org/0000-0002-1026-7476
Fabien Zoulim http://orcid.org/0000-0002-2245-0083

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