Kinetics of serum O‐glycosylated M‐hepatitis B surface antigen with hepatocellular carcinoma history and nucleos(t)ide analogue therapy in hepatitis B patients

A newly developed O‐glycosylated M‐hepatitis B surface antigen (HBsAgGi) measurement system can detect hepatitis B surface antigen (HBsAg) associated with infectious particles. We investigated the association of HBsAgGi levels with clinical parameters and a history of hepatocellular carcinoma (HCC) development in a cross‐sectional cohort analysis (Study 1) as well as the quantitative changes in HBsAgGi during nucleos(t)ide analogue (NA) therapy in a longitudinal cohort analysis (Study 2). A total of 124 patients with genotype C chronic HBV infection were analysed in Study 1 to evaluate correlations of HBsAgGi with conventional HBV markers and HCC history. Among those, 36 patients receiving NA therapy were enrolled in Study 2 for quantitative comparisons between pre‐treatment baseline and 48 weeks of NA therapy. In Study 1, serum HBsAgGi was significantly associated with HBsAg (r = .5857, p < .00001) and weakly but significantly correlated with HBV DNA (r = .2936, p = .001). Although HBsAgGi (p = .111) was comparable between HCC history (+) group and HCC history (−) group, the HBsAgGi/HBsAg ratio (p = .011) was significantly higher in HCC history (+) patients. In Study 2, HBsAgGi was significantly decreased after 48 weeks of NA therapy (p < .001). HBsAg findings were similar (p = .005) along with an HBV DNA reduction (p < .001). In the baseline hepatitis B e antigen (HBeAg) (+) subgroup, HBsAgGi decreased significantly between baseline and 48 weeks of NA (p = .005), while HBsAg was comparable (p = .051). Low HBsAg and high HBsAgGi were associated with a history of HCC development. HBsAgGi decreased significantly by 48‐week NA therapy.


| INTRODUC TI ON
Hepatitis B virus (HBV) infection is a global health problem, with frequent morbidity and mortality in afflicted patients. It is estimated that more than 350 million people worldwide are infected with HBV, causing roughly 600,000 deaths yearly. 1 HBV infection outcomes vary from spontaneous clearance to viral persistence that may progress to liver cirrhosis and hepatocellular carcinoma (HCC). 2,3 As such, serological biomarkers are needed to accurately evaluate disease status.
In terms of the biomarkers associated with HBV infection, hepatitis B surface antigen (HBsAg), HBV DNA and hepatitis B core-related antigen (HBcrAg) levels have been used in the clinical setting to estimate HBV replication activity, predict the therapeutic response to nucleos(t)ide analogue (NA) therapy, and monitor for HCC development. 4 There are three types of HBsAg on the HBV envelope protein: small proteins of hepatitis B virus surface antigen (S-HBsAg), middle proteins of hepatitis B virus surface antigen (M-HBsAg) and large proteins of hepatitis B virus surface antigen (L-HBsAg). Each variant plays a different role, with L-HBsAg encapsulating the HBV core. 2,5 Structurally, the amino-terminal preS1 domain is present only in L-HBsAg, the preS2 domain is found in both L-HBsAg and M-HBsAg and the S domain is common to L-HBsAg, M-HBsAg and S-HBsAg. 6 HBV particles in a patient's blood include infectious virions called Dane particles containing viral DNA as well as non-infectious particles called subviral particles (SVPs). At 1000 times more abundant than Dane particles, SVPs are composed mainly of S-HBsAg proteins, while the virions essential for HBV infection are composed mainly of M-HBsAg proteins and partially of L-HBsAg proteins. 7 In order to accurately evaluate disease status, it is therefore important to monitor HBV virions and distinguish them from SVPs.
Although the currently available quantitative HBsAg assay has been proven to correlate with serum HBV DNA and intrahepatic covalently closed circular DNA (cccDNA) levels, 8

it cannot distinguish
HBsAg in terms of HBV virions and SVPs. In order to monitor HBV virions, a newly developed method to specifically measure HBsAg associated with Dane particles rather than non-infectious SVPs has become available. Recent glycotechnology results revealed that the preS2 domain was present in M-HBsAg and L-HBsAg, the former containing an O-glycan and preS2 but the latter not, specifically in genotype C HBV infection. 9 Accordingly, a recombinant monoclonal antibody that specifically recognizes O-glycosylated M-HBsAg (HBsAgGi) has been made 9 and is now commercially available.
This study investigated the association of HBsAgGi with clinical parameters and HCC development using a cross-sectional cohort analysis (Study 1) in addition to the quantitative changes in HBsAgGi during NA therapy in a longitudinal cohort analysis (Study 2).

| Measurement of HBsAgGi levels by enzyme-linked immunoassay (ELISA)
HBsAgGi levels were measured by a commercially available ELISA kit (RCMG Inc) according to the manufacturer's instructions 9 using patient serum samples immediately stored at −20°C after collection.
Sample absorbance at 450 nm was measured using an ELISA plate reader. HBsAgGi levels were calculated based on a standard curve and dilution factor. The ELISA kit has a measure range of 10-200 ng/ mL and samples with serum levels over 200 ng/mL were diluted and re-assayed to determine their levels accurately. In Study 1, HBsAgGi levels were measured using serum samples when patients visited the hospital at the first time point in 2021. In Study 2, HBsAgGi levels were quantified at a baseline point just before NA commencement (baseline in Table 3 and in Figure 3) and at 48 weeks of NA therapy (48 weeks in Table 3 and in Figure 3).

| Definition of a history of HCC development
Patients with HCC were defined as having a history of HCC, that is, HCC history (+), by the end of 2021 or complicating HCC in 2021.
HCC was diagnosed by imaging characteristics, arterial hypervascularity and venous or delayed phase washout by contrast-enhanced dynamic computed tomography and/or magnetic resonance imaging when a nodular lesion was detected by ultrasonography or a tumour marker was elevated. CH, HBsAg positive for more than 6 months, serum HBV DNA greater than 3.3 log IU/mL, persistent or intermittent elevation of ALT or aspartate aminotransferase (AST) levels; and LC, histological diagnosis (METAVIR F4) 11 or clinical diagnosis (blunted nodular liver edges on abdominal ultrasound scan ± splenomegaly + platelet count below 15 × 10 4 /μL).

| Ethics
This study was reviewed and approved by the Institutional Review An opt-out system is in place at our institution. All information on the protocol and conduct of the study, including its purpose, is available on the Department of Medicine, Shinshu University School of Medicine website (http://www.shins hu-u.ac.jp/facul ty/ medic ine/chair/ i-2nai/). If patients do not wish to participate in the research, they are freely able to opt out of the study.

| Statistical analysis
Statistical analysis and data visualization were carried out using StatFlex ver. 7.0.11 software (Artech Co., Ltd). Continuous baseline data are expressed as the median and interquartile range and statistically evaluated by means of the Mann-Whitney U test.
Categorical variables are presented as the frequency (percentage) and analysed using the chi-square test. The Wilcoxon signed rank test was employed to analyse the differences in continuous variables between the immediately pre-treatment baseline and 48 weeks of NA time points. To determine the independent factors associated with HCC history, multivariate logistic regression analysis was performed using the following indices: age, sex, platelet count, albumin, total bilirubin, AST, ALT, HBV DNA, HBeAg (positive or negative), HBsAg, HBsAgGi and HBsAgGi/ HBsAg ratio (four divided groups). All statistical tests were twosided and evaluated at the 0.05 level of significance.

| Patient characteristics of all subjects in Study 1
The cohort's characteristics in Study 1 are summarized in Table 1

| Baseline correlations of HBsAgGi with HBsAg and HBV DNA (Study 1)
At baseline, median serum HBsAg and HBsAgGi levels were 2.90 log IU/mL and 3.41 log ng/mL, respectively. Median serum HBsAgGi levels for AC, IC, CH and LC were 4.06, 3.76, 3.40 and 3.26 log ng/ mL, respectively, with no significant differences among the groups ( Figure S2). HBsAgGi was significantly correlated with HBsAg (r = .5857, p < .00001) ( Figure 1A) and significantly but weakly cor-

| Comparisons of clinical characteristics between HCC history (−) and HCC history (+) groups (Study 1)
Clinical characteristic comparisons between the HCC history (−) and HCC history (+) groups are presented in Table 1 confidence interval: 1.34-13.12, p = .014) as an independent factor associated with HCC history (+) ( Table 2). Area under the ROC curve (AUROC) analysis was performed for HBsAgGi/HBsAg ratio predicting HCC history and revealed a cutoff value for HBsAgGi/HBsAg ratio of 1.37 as calculated by the Youden index. In this setting, sensitivity and specificity were 83% and 50%, respectively ( Figure S3).

| Characteristics of the 36 patients receiving NA therapy in Study 2
Of the 124 patients in Study 1, 36 patients had received NA therapy for 48 weeks. The characteristics of the cohort in Study 2 are TA B L E 1 Clinical comparisons between HCC (−) and HCC (+) groups.

| Changes in serum HBsAg and HBsAgGi during NA therapy (Study 2)
HBsAg levels (from 3.55 to 3.39 log IU/L, p = .005) and HBsAgGi levels (from 4.48 to 3.55 log ng/mL, p < .001) decreased significantly from baseline to 48 weeks ( Figure 3A and Figure 3B). In the subgroup of HBeAg (+) patients, HBsAg levels were comparable (from 3.79 to 3.48 log ng/mL, p = .051) ( Figure 3C), while HBsAgGi levels decreased significantly (from 4.81 to 3.79 log ng/mL, p = .005) ( Figure 3D) during NA treatment. In the subgroup of HBeAg (−) patients, HBsAg levels decreased significantly from baseline to 48 weeks (from 3.36 to 3.14 log ng/mL, p = .021) ( Figure 3E), with HBsAgGi levels remaining comparable (from 3.40 to 3.16 log ng/mL, p = .092) ( Figure 3F). Chronic HBV infection has been associated with an increased risk of HCC. 15 Since HCC can develop even in patients with chronic HBV but without cirrhosis, it is clinically important to predict HCC onset on a patient-by-patient basis. In addition to disease progression to cirrhosis, several prognostic HCC factors have also been revealed to date, including high HBV DNA level, high HBsAg level, male gender and family history of HCC. 16 Genotype C infection is an independent risk factor for HCC as well. 17 As the majority of chronic HBV infection in Japan is genotype C, predicting HCC development in HBV patients with this genotype is clinically important. HCC development. 16,18 Our earlier results showed that low HBsAg levels were associated with a history of HCC in a cross-sectional analysis, 19 which might have been influenced by higher cohort age.

| DISCUSS ION
However, previous studies showed that preS/S mutations in the HBV genome could result in impaired extracellular secretion of HBsAg produced by hepatocytes. 20,21 Another report described that patients with preS/S mutations often had histological advancement of the liver despite low-serum HBsAg levels. 22   changes. 14 Longitudinal studies are required to clarify the utility of HBsAgGi for predicting relapse during NA therapy and determining when to discontinue NA treatment, just as for the reported usefulness of HBcrAg. 27,28 This study had several limitations. First, it was conducted at a single center with a retrospective design. Second, the number of patients was relatively small. Third, the observation period was short in comparisons between baseline and 48 weeks of NA treatment.
Larger multi-centre analyses are needed to uncover the clinical utility of HBsAgGi.

| CON CLUS IONS
In cross-sectional analysis, HBsAgGi was significantly correlated with HBsAg and HBV DNA, with low HBsAg and high HBsAgGi associated with HCC development in genotype C chronic HBV patients. Longitudinal analysis revealed that HBsAgGi decreased significantly by 48-week NA therapy. Prospective longitudinal studies are warranted to determine the applicability of HBsAgGi as a serological viral marker.

ACK N O WLE D G E M ENTS
We thank Yuki Akahane and Asami Yamazaki for their technical assistance as well as Trevor Ralph as the Senior Editor of Impact Language Services for his English editorial assistance.

FU N D I N G I N FO R M ATI O N
The authors sincerely appreciate the research support provided in part by the Japan Agency for Medical Research and Development (AMED) (JP21fk0210084, JP22fk0210112).

CO N FLI C T O F I NTER E S T S TATEM ENT
All authors have nothing to disclose regarding funding from industries or other conflicts of interest with respect to this manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.