Early Serum HBV RNA Level in Combination With the HBeAg Response Can Effectively Predict the HBeAg Response in Patients on Nucleos(t)ide Analogue Therapy (ClinicalTrials.gov

Background: Serum HBV RNA level has the potential to monitor antiviral therapy in patients with chronic hepatitis B. This study aimed to explore serum HBV RNA dynamic change pattern and its predict value on the ecacy of 96 weeks nucleos(t)ide analogue (NA) treatment in HBeAg-positive and HBeAg-negative patients with chronic hepatitis B (CHB). Methods: A real-life cohort study of 78 patients with CHB on NA treatment was conducted. Dynamic change patterns of serum HBV RNA and correlation with other HBV markers in the early treatment period of 96 weeks of NA treatment in patients with CHB were determined and compared. The performance of serum HBV RNA on treatment ecacy was analyzed by receiver operating characteristic (ROC) analyses. Results: HBeAg-positive and HBeAg-negative patients with CHB had similar viral change patterns during NA treatment. Serum HBV RNA level was consistently correlated with HBeAg and HBsAg titers in HBeAg-positive patients during NA treatment, but serum HBV RNA was only moderately correlated with serum HBV DNA level in HbeAg-negative patients before treatment. Serum HBV RNA decreased more rapidly in patients with the HBeAg seroconversion (SC) response than in patients without the HBeAg SC response; it had good early discriminatory ability for the HBeAg response with area under the ROC curve (AUROC) of 0.70 and 0.730 at 12 and 24 weeks of treatment, respectively. The cutoff value of serum HBV RNA of 4.31 log cps/mL in combination with the HBeAg decrease degree of 1.55 at 24 weeks of treatment had a good two-way predictive capability for the HBeAg response (PPV%: 83.33% and NPV%: 81.25%), and the specicity was 96.30%. Conclusion: Serum HBV RNA level had early discriminatory ability for the HBeAg response. Early HBeAg response can improve the discriminatory ability of serum HBV RNA. in CHB patients on NAs therapy by HBeAg status. Correlation between serum HBV RNA and other HBV indicators were analyzed using Spearman analysis. Number


Introduction
Long-term nucleos(t)ide analogue (NA) treatment can improve liver in ammation, ameliorate the development of liver brosis, and reduce the risk of hepatocellular carcinoma (HCC) through e cient suppression of hepatitis B virus (HBV) replication. However, given the limited effect on covalently closed circular DNA (cccDNA), NA therapy rarely leads to a functional cure (de ned as hepatitis B surface antigen [HBsAg] loss with undetectable HBV DNA) [1][2][3]. HBeAg loss and seroconversion (SC) are associated with immune restoration and can be the impetus for HBsAg loss and SC [1,2,4,5].
Therefore, to improve treatment responses, pretreatment and on-treatment viral factors must be assessed.
Several HBV biomarkers, including HBV DNA, HBsAg titer, and hepatitis B core-related antigen (HBcrAg), have been used to monitor HBV chronic infection and antiviral therapy [6][7][8][9][10]. Unfortunately, these indicators are currently unable to effectively predict or guide NA therapy alone. Serum HBV RNA, a new surrogate for intrahepatic cccDNA transcription, has the potential to monitor chronic HBV infection, and a growing body of research suggested that serum HBV RNA may be a useful marker for monitoring chronic HBV infection including natural history, treatment, and prognosis [11][12][13][14][15][16][17][18][19]. However, previous research yielded inconsistent results, such as the correlation between serum HBV RNA and other viral markers in HBeAg-positive and HBeAg-negative patients with or without antiviral treatment [19][20], early serum HBV RNA kinetics in patients on NA therapy [21], and the potential use of serum HBV RNA and the cutoff value of serum HBV RNA level for prediction [22].
In this work, we aimed to determine the early kinetics of serum HBV RNA in HBeAg-positive and HBeAg-negative patients and evaluate the performance of early-stage serum HBV RNA for monitoring NA therapy patients with chronic hepatitis B (CHB).

Methods And Materials
Study design A real-life cohort study was conducted in Shanghai Changhai Hospital between February 12, 2016 andFebruary 21, 2018. Dynamic change patterns of serum HBV RNA in different patients with CHB undergoing long-term antiviral treatment were determined and compared. Furthermore, the role of serum HBV RNA alone or in combination with other HBV biomarkers on the treatment e cacy of HBeAg-positive patients with CHB was explored. The study was approved by the ethics research committee (CHEC2019-056) of Shanghai Changhai Hospital and registered on ClinicalTrials.gov (NCT03909191). Written informed consent was obtained from each patient.

Patients
Patients enrolled met the following criteria: positive for HBsAg more than 6 months before enrollment; alanineamino transferase (ALT) level higher than the upper limit of detection (14 days interval); no decompensated cirrhosis; no hepatitis C, hepatitis D, or human immunode ciency virus co-infection; no HCC and other tumors or history of severe hepatitis; and no other systemic diseases, such as a history of cardiopulmonary diseases, thyroid disorders, immune system disorders, epilepsy, or mental illness (such as severe depression). The patients enrolled in this study were treated with rst-line NAs or Peg interferon (IFN), followed by a switch to NAs for 96 weeks.

Antiviral response de nition
HBeAg SC was de ned as the loss of HBeAg and the appearance of anti-HBeAg on two occasions, at least one month apart in HBeAg-positive patients during 96 weeks of NA treatment.
For subsequent analyses, HBeAg-positive patients who achieved HBeAg SC and those who did not achieve HBeAg seroconversion (NSC) were divided into the SC and NSC groups, respectively.

Serological and virological detection
Serum HBV RNA quanti cation Serum HBV RNA was detected by RNA simultaneous ampli cation (SAT) using the commercially available HBV-SAT kit (Rendu Biotechnology, Shanghai, China) as previously described [11,12]. Primers and probes were designed to amplify an HBV pre-genomic RNA (pgRNA) conserved region [23]. RNA extraction, ampli cation, and detection were processed on an automated AutoSAT system (Rendu Biotechnology, Shanghai, China). The HBV RNA assay was calibrated using an armored HBV RNA standard traceable to the HBVRNA standard in vitro transcripts. The minimum detection limit (MDL) was 100 copies/mL.

Serum HBV DNA quanti cation
Serum HBV DNA was quanti ed by the commercially available hepatitis B viral DNA Determination Kit (Sansure Biotech, China), with a detection range of 1×10 2 IU/mL to 5×10 8 IU/mL. The value below the detection limit was replaced with the low detection limits.

HBV serological test
Serum HBsAg, HBeAg, anti-HBs, anti-HBe, and anti-HBc were measured by chemiluminescence microparticle immunoassays using the Architect i2000SR platform and Abbott Architect reagents (Abbott Laboratories, North Chicago, IL, USA) in our clinical laboratory.

Statistical analysis
The data are presented as the mean, median (interquartile range) or proportion based on data type and distribution. The two groups (SC vs. NSC and HBeAg-positive vs. HbeAg-negative CHB) were compared using the Mann-Whitney U test for continuous data and chi-squared test for categorical data. Spearman correlation coe cients ® were used to compare the correlation between two variables. Logistic regressions were used to assess the odds ratio and 95% con dence interval (95% CI) of HBV markers. Receiver operating characteristic (ROC) curves were used to compare the relative sensitivity and speci city of HBV RNA, HBsAg, HBeAg, and HBV DNA as predictors of antiviral e cacy. The cutoff value was chosen based on the ROC curve when the sensitivity and speci city were both relatively high. A P-value (two-tailed) of 0.05 was At baseline, HBeAg-positive patients with CHB had signi cantly higher viral replication level compared with HBeAg-negative patients with CHB. However, during the 96 weeks of NA treatment, HBeAg-positive and HBeAg-negative patients achieved similar serum HBV DNA below the MDL (< MDL), HBsAg loss rate, and ALT normalization rate (Table 1). Of these 79 patients with CHB, only one patient achieved HBsAg loss, whereas four patients (two HBeAg-positive and two HBeAgnegative patients with CHB) achieved HBsAg < 100 IU/mL during the 96 weeks of antiviral treatment. Fourteen (25.93%) HBeAg-positive patients with CHB achieved SC response. HBeAg-positive patients with SC had signi cantly higher ALT normalization rate and serum HBV DNA < MDL rate than NSC patients (Table 1).    Table 2). The 24-week HBeAg cutoff value of 0.306 log 10 S/CO showed a two-way predictive capability for the HBeAg response (PPV% and NPV% both > 80%; Table 2). In addition to the absolute cutoff value of HBeAg, the titer decreases from baseline with a cutoff value of 1.55 log IU/mL at 24 weeks of treatment had a good NPV% (86.47%) and high speci city (86.49%) (Table S1). To nd better predictors for the HBeAg response, we explored the predictive value of serum HBV RNA in combination with other classical HBV biomarkers and found that serum HBV RNA combined with HBeAg had good predictive value and high AUROC scores (0.854, 95% CI: 0.754-0.954; Table S2). The absolute cutoff value of serum HBV RNA of 4.31 combined with HBeAg decrease degree of 1.55 at 24 weeks of treatment had a good two-way predictive capability for the HBeAg response (PPV%: 83.33% and NPV%: 81.25%), and the speci city was 96.30% (Table S2).

Discussion
Here, we longitudinally studied the dynamic pattern of serum HBV RNA pro les in HBeAg-positive or HBeAg-negative patients with CHB subjected to NA treatment. This work aimed to reveal whether serum HBV RNA has similar value in monitoring the antiviral response in HBeAg-positive and -negative patients. We found that the dynamic change pattern of serum HBV RNA in HBeAg-positive patients on long-term NA treatment mirrored that of serum HBV DNA, HBsAg, and HBeAg. Serum HBV RNA sustained moderate to strong correlation with these viral markers. However, the correlation between serum HBV RNA and other HBV biomarkers was not sustained in HBeAg-negative patients with CHB during NA treatment. These results indirectly illustrated that serum HBV RNA quanti cation may play a potential monitoring value on the NA treatment response in HBeAg-positive patients with CHB, other than in HBeAg-negative patients.
As a surrogate biomarker for intrahepatic cccDNA quantity and transcript activity, serum HBV RNA level has been con rmed to be a valuable marker for CHB management, including natural course management and antiviral management [10-16, 18-20, 24-27]. Our previous study and other researches revealed that serum HBV RNA alone or combined with other viral indicators such HBsAg titer or HBcrAg level can guild antiviral cessation in patients with CHB [12,15,16,28]. In this study, we further studied the monitoring value of serum HBV RNA on the NA treatment response. We found no difference in serum HBV RNA level at baseline between patients with the SC response or not, but serum HBV RNA level in patients with the SC response was obviously lower than that in patients without the SC response at the early stage of NA treatment. Moreover, the AUROC values of serum HBV RNA at 12 and 24 weeks of treatment exceeded 0.70, indicating that serum HBV RNA level had early predictability of the HBeAg SC response in patients on NA treatment. We then analyzed the absolute cutoff value of serum HBV RNA at 12 and 24 weeks of treatment separately and found that the cutoff of HBV RNA at 4.94 or 4.31 log10 copies/mL at 12 and 24 weeks of treatment, respectively, had the highest NPV for the SC response in NA treatment. All these results con rmed that serum HBV RNA alone showed good discriminatory ability for the antiviral response on NA treatment. Compared with HBeAg level and HBsAg titer of the same time point, serum HBVRNA alone had no obvious advantage in predicting the antiviral response. Further analysis of the NA treatment response of HBV biomarkers demonstrated no obvious advantage of serum HBV RNA in predicting the SC response during 96 weeks of NA treatment. However, serum HBV RNA value combined with HBeAg decrease degree at 24 weeks of NA treatment showed a good predictive value for the SC response, with high speci city and AUROC value. These results were similar to the ndings reported by Wang[18] and Luo [29], who both found that the combination of serum HBV RNA and HBeAg response shows better predicting value for NA treatment response than serum HBV RNA alone.
Although our results here and the ndings of previous studies revealed that serum HBV RNA alone or in combination with HBeAg treatment response can monitor NA treatment early, the cutoff values of serum HBV RNA in the literature varied widely [16,29]. This difference may be related to the methodological heterogeneity of serum HBV RNA detection. Given the complexity of serum HBV RNAs, serum HBV RNA levels detected by different primers targeting varying regions of the HBV genome were not only inconsistent [30] but also different in treatment outcomes [31]. Therefore, before serum RNA is applied to clinical practice in the future, further understanding of the biological signi cance of serum HBV pgRNA and standardization of testing methods is critical.
As a real-life cohort study, our results were more representative of the clinical reality than team-registered studies. However, our study had some shortcomings, such as a relatively small sample size, and the signi cance of serum HBV RNA prediction needs to be further veri ed in larger sample sizes. Moreover, in our study, HBeAg was measured using a chemiluminescence microparticle immunoassay, which is the most common assay method in China but not a total quantitative test. Despite these limitations, the HBeAg level still has meaningful reference value in clinical practice.

Conclusion
In