Albumin-bilirubin score is associated with response to pegylated interferon and nucleos(t)ide analogues in chronic hepatitis B patients

Clinica Chimica Acta 502 (2020) 120–127

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Albumin-bilirubin score is associated with response to pegylated interferon and nucleos(t)ide analogues in chronic hepatitis B patients

T

Zhen Xuna,b,d,1, Can Liub,c,d,1, Qing-Qing Yua,b,d, Jin-Piao Linb,c,d, Jin-Lan Huangb,c,d, ⁎ Ting-Wen Yanga, Wen-Nan Wub,c, Song-Hang Wub,c, Qi-Shui Oub,c,d, a

First Clinical College, Fujian Medical University, Fuzhou, China Department of Laboratory Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China c Gene Diagnostic Laboratory, The First Affiliated Hospital of Fujian Medical University, Fuzhou, China d Fujian Key Laboratory of Laboratory Medicine, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Albumin-bilirubin Chronic hepatitis B Pegylated interferon Nucleos(t)ide analogues Natural history Treatment response

Background and aim: Recently, the role of albumin-bilirubin (ALBI) score in chronic hepatitis B (CHB) has not been well-understood. We aimed to investigate the association of ALBI score with natural history of chronic HBV infection and treatment response of CHB patients. Methods: The ALBI score in a cohort of 849 individuals including 721 chronic HBV-infected patients naïve to anti-HBV treatment in different phases and 128 healthy controls were estimated. Additionally, the dynamic changes of ALBI score of 243 hepatitis B e antigen (HBeAg)-positive CHB patients treated with pegylated interferon-alpha (PEG-IFN-α) or nucleos(t)ide analogues (NAs) were tested for 72 weeks. Results: ALBI score differed among phases, with the highest score in HBeAg-positive CHB patients, followed by HBeAg-negative CHB patients, HBeAg-positive chronic HBV infection, and HBeAg-negative chronic HBV infection. Besides, CHB patients harbouring high baseline ALBI score exhibited a relatively stronger therapeutic response to PEG-IFN-α or NAs. Moreover, the rate of HBeAg and HBsAg loss in patients with ALBI grade 2 was persistently higher than that in patients with ALBI grade 1 throughout the course of treatment. Furthermore, ALBI score was an independent predictor of sustained response achievement. The combined use of ALBI score, HBeAg and ALT could enhance the predictive value of treatment response. Conclusions: ALBI score differed significantly across the natural course of chronic HBV infection and was correlated with PEG-IFN-α and NAs treatment response in HBeAg-positive CHB patients, which suggested that ALBI score could be useful as an auxiliary clinical factor to determine the initiation of therapy and predict stronger antiviral treatment response.

1. Introduction Chronic HBV infection remains a serious public health issue, affecting more than 240 million people worldwide [1,2]. The prevention and control situation of chronic hepatitis B (CHB) are still grim, and the realization of CHB precision diagnosis and treatment has vital economic and social significance [3,4]. Currently, the natural history of chronic HBV infection is classified

into four phases (HBeAg-positive chronic HBV infection, HBeAg-positive chronic hepatitis B, HBeAg-negative chronic HBV infection and HBeAg-negative chronic hepatitis B) dependent on clinical laboratory assessment of biochemical, immunological and virological characteristics [5]. Among them, HBeAg-positive CHB and HBeAg-negative CHB patients have poor liver function. Although serial monitoring of hepatitis B e antigen (HBeAg), HBV DNA and alanine aminotransferase (ALT) levels (determined the disease phase of an individual chronic

Abbreviations: ALBI, albumin-bilirubin; CHB, chronic hepatitis B; HBeAg, hepatitis B virus e antigen; PEG-IFN-α, pegylated interferon-alpha; NAs, nucleos(t)ide analogues; SR, sustained response; NR, nonresponse; HBV, hepatitis B virus; ALT, alanine aminotransferase; HBsAg, hepatitis B virus surface antigen; HCC, hepatocellular carcinoma; TBIL, total bilirubin; ALB, albumin; DBIL, direct bilirubin; IBIL, indirect bilirubin; TP, total protein; GLO, globin; AST, aspartate amino transferase; GGT, gamma-glutamyltransferase; LDH, lactic dehydrogenase; ALP, alkaline phosphatase; CHE, cholinesterase; Anti-HBe, hepatitis B virus e antibody; Anti-HBc, hepatitis B virus core antibody; BMI, body mass index ⁎ Corresponding author at: 20 Chazhong Road, Fuzhou 350005, Fujian, China. E-mail address: [email protected] (Q.-S. Ou). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.cca.2019.12.020 Received 4 September 2019; Received in revised form 23 December 2019; Accepted 24 December 2019 Available online 28 December 2019 0009-8981/ © 2019 Elsevier B.V. All rights reserved.

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continued for 48 weeks and were followed by a 24-week post-treatment observation period. According to curative effects at 24 weeks posttreatment (week 72), HBeAg-positive CHB patients were divided into sustained response (SR) group and non-response (NR) group. SR to treatment was defined according to the EASL guidelines [5] as HBeAg < 1 S/CO, HBV DNA < 500 IU/ml and ALT < 40 U/L when followed-up at 24 weeks post-treatment (week 72). Patients were deemed to have NR to therapy if they did not meet all of the criteria above.

HBV patient and the indication for antiviral treatment) are required in most cases, some subjects fall into an indeterminate grey area and result in the dilemma in antiviral therapy [6,7]. On the other hand, the therapeutic goal of CHB is to continuously inhibit HBV replication and control inflammation [8]. Currently, interferons and nucleos(t)ide analogues (NAs) are widely used in clinical treatment for CHB [9]. However, the response to pegylated interferon-a (PEG-IFN-α) or NAs therapy varies widely. Long-term use of NAs can induce resistance and very low HBeAg seroconversion rate [10]. Besides, only 3–11% of HBeAg-positive CHB patients who received IFN-α have hepatitis B surface antigen (HBsAg) clearance [11,12], and 29–32% of HBeAg seroconversion occurs after a 48-week course of treatment [13]. Therefore, if it is possible to predict whether CHB patients are suitable for treatment, it will reduce the economic expenditure of patients. So far, there is still no ideal indicator to accurately predict the efficacy of antiviral drugs [14]. For newly diagnosed patients, it is even more difficult to determine the therapeutic effect before administration. Therefore, these limitations suggest that we should look for new indicators for predicting the therapeutic effect of antiviral drugs from other perspectives. Albumin-bilirubin (ALBI) score has been initially used for evaluating the severity of liver dysfunction in patients with hepatocellular carcinoma (HCC) [15]. The score involves only two common laboratory parameters of total bilirubin (TBIL) and albumin (ALB). Currently, it has been confirmed that ALBI has a significantly prognostic performance for long-term survival [16] and tumor recurrence [17] predictions in HCC patients treated with surgical resection. Besides, ALBI score is also an independent prognostic factor of primary biliary cirrhosis patients [18]. Recently, a retrospective study demonstrated that ALBI grade was effective in predicting the long-term prognosis for patients with HBV-related cirrhosis [19]. Although ALBI score is initially used for evaluating liver function reserve in patients with HBV-related cirrhosis or HCC, most CHB patients also have abnormal liver function [20]. As the main cause of liver cirrhosis and HCC, there are still little data about ALBI in CHB patients. The distribution of ALBI score in each phase of CHB has not been fully explored. Moreover, whether ALBI score play any role in influencing the therapeutic efficacy of anti-HBV treatment remains unknown. In this study, ALBI score of 849 individuals (721 chronic HBV-infected patients naïve to anti-HBV treatment in different phases and 128 healthy controls) and the dynamic changes of ALBI score in 243 HBeAgpositive CHB patients receiving antiviral therapy were tested. We aimed to explore the ALBI score in each phase of HBV natural history and HBeAg-positive CHB patients receiving PEG-IFN-α or NAs treatment to investigate the association of ALBI score with natural progression of chronic HBV infection and long-term outcome under treatment of CHB patients.

2.2. Laboratory measurement of clinical indicators The clinical liver function indicators measured for this study included TBIL, direct bilirubin (DBIL), indirect bilirubin (IBIL), total protein (TP), ALB, globin (GLO), ALT, aspartate amino transferase (AST), gamma-glutamyltransferase (GGT), lactic dehydrogenase (LDH), alkaline phosphatase (ALP) and cholinesterase (CHE). All of the clinical indicators were quantified in samples taken at baseline, during the treatment period and follow-up periods by an automatic biochemical analyser ADVIA 2400 (Siemens, Munich, Germany). Sampling was performed at weeks 0, 4, 12, 24, 36, 48, 60 and 72 (week 0, namely baseline, was the time point at which treatment began). HBV DNA was detected by quantitative real-time PCR method (Sansure Biotech Inc., Hunan, China) and Roche Lightcycler 480 (Roche Corporation, Basel, Switzerland). HBsAg, HBeAg, hepatitis B e antibody (anti-HBe) and anti-HBc were quantified using an automated chemiluminescent microparticle immunology analyser (Abbott I2000, Abbott Laboratories, Chicago, IL, USA). 2.3. Calculation of score The ALBI score was calculated from two variables including bilirubin and albumin. ALBI score = 0.66 × log10 (TBIL μmol/L) − 0.085× (albumin g/L) [15,17,21]. Patients were divided into three groups as grade 1 (≤−2.60), grade 2 (> −2.60, ≤−1.39) and grade 3 (> −1.39) according to ALBI score [15,17,21]. There were no ALBI grade 3 patients in the cohort because they were mostly diagnosed with decompensated liver cirrhosis or liver cancer. 2.4. Statistical analysis Data were analyzed using the SPSS version 20 software (SPSS, IBM, Chicago, IL, USA). Statistical comparisons between two groups were performed using the two-tailed Student’s t-test if quantitative variables were normal and homogeneous distribution or Mann-Whitney U test if not. The χ2 test was used to examine the differences for categorical variables. Univariate and multivariate logistic regression analyses were carried out to identify independent factors for SR. Differences of all tests achieving P-values < 0.05 were considered to be statistically significant.

2. Materials and methods 2.1. Patients A total of 849 subjects were recruited at the First Affiliated Hospital of Fujian Medical University, including 721 chronic HBV-infected patients naïve to anti-HBV treatment and 128 healthy controls. According to the natural history of chronic HBV infection, patients were divided into four phases [5]: HBeAg-positive chronic HBV infection, HBeAgpositive chronic hepatitis B (HBeAg-positive CHB), HBeAg-negative chronic HBV infection and HBeAg-negative chronic hepatitis B (HBeAgnegative CHB). A summary of patient characteristics is presented in Table 1. All patients provided written informed consent and this research was approved by the Ethics Committee of the First Affiliated Hospital of Fujian Medical University (Grant No. FY2013017). Among HBeAg-positive CHB patients, a total of 243 patients who had received anti-HBV treatment by PEG-IFN-α or NAs for 48 weeks were enrolled (Supplementary Table 1). Therapeutic regimens

3. Results 3.1. Clinical characteristics of the study population For the liver function profile of chronic HBV infection, a cohort of 1522 treatment-naïve patients with chronic HBV infection underwent series of examinations. Among them, 801 patients were excluded for some reasons (Fig. 1). Baseline demographics and clinical characteristics of the entire 721 patients and 128 healthy controls were shown in Table 1. Owing to the stringent definition criteria, differences in ALT, HBsAg, HBeAg and HBV DNA levels were observed between the natural progression of chronic HBV infection (Table 1). 121

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Table 1 Baseline demographics and clinical characteristics of the study population. Variables

Healthy control

HBeAg-positive chronic HBV infection

HBeAg-positive chronic hepatitis B

HBeAg-negative chronic HBV infection

HBeAg-negative chronic hepatitis B

Number of participants

128

238

258

128

97

Clinical parameters Gender, n Male/n Female Age, years

77/51 29 ± 6

118/120 31 ± 7

178/80 30 ± 6

82/46 36 ± 8

75/22 42 ± 9

Laboratory parameters Log10 HBV DNA, IU/ml Log10 HBsAg, IU/ml Log10 HBeAg, S/CO Anti-HBe, S/CO Anti-HBc, S/CO Total bilirubin, μmol/L Direct bilirubin, μmol/L Indirect bilirubin, μmol/L Total protein, g/L Albumin, g/L Globin, g/L ALT, U/L AST, U/L ALT/AST GGT, U/L LDH, U/L ALP, U/L Cholinesterase, U/L

– – – – – 14.4 ± 5.3 4.5 ± 1.6 9.9 ± 3.4 74.9 ± 4.1 47.2 ± 3.9 28.1 ± 3.0 21 ± 11 21 ± 5 1.0 ± 0.4 21 ± 10 183 ± 28 66 ± 22 7766 ± 1821

7.6 ± 0.6 4.6 ± 0.3 3.1 ± 0.2 51.4 ± 9.8 7.6 ± 2.0 12.9 ± 5.2 4.0 ± 1.4 8.9 ± 3.9 73.2 ± 4.2 44.5 ± 2.9 29.1 ± 4.5 29 ± 12 23 ± 7 1.3 ± 0.4 19 ± 12 181 ± 31 67 ± 18 7955 ± 1767

7.3 ± 1.0 4.1 ± 0.6 2.8 ± 0.5 37.4 ± 22.2 10.5 ± 3.9 14.9 ± 6.0 5.6 ± 3.0 9.4 ± 3.7 72.5 ± 5.2 43.4 ± 3.5 29.0 ± 4.3 189 ± 117 99 ± 65 2.0 ± 0.8 62 ± 59 194 ± 56 79 ± 31 7821 ± 1883

2.5 ± 0.5 2.5 ± 1.1 −0.5 ± 0.3 0.1 ± 0.2 9.4 ± 1.0 12.3 ± 4.2 4.5 ± 5.7 8.2 ± 2.9 74.7 ± 4.0 46.2 ± 2.8 28.6 ± 2.7 27 ± 10 23 ± 6 1.2 ± 0.4 23 ± 16 176 ± 27 66 ± 19 9515 ± 2268

5.4 ± 1.3 3.2 ± 0.8 −0.4 ± 0.2 0.3 ± 1.4 10.3 ± 2.7 14.5 ± 5.4 5.3 ± 2.1 9.2 ± 3.8 74.9 ± 4.8 44.7 ± 3.1 30.2 ± 3.9 132 ± 76 81 ± 66 1.9 ± 0.7 63 ± 54 198 ± 65 78 ± 32 8108 ± 1826

ALBI score

−3.24 ± 0.23

−3.10 ± 0.26

−2.86 ± 0.39

−3.23 ± 0.24

−2.96 ± 0.38

Data are mean ± SD unless otherwise indicated. HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; anti-HBe, hepatitis B e antibody; anti-HBc, hepatitis B core antibody; ALT, alanine aminotransferase; AST, aspartate amino transferase; GGT, gamma-glutamyltransferase; LDH, lactic dehydrogenase; ALP, alkaline phosphatase; ALBI, albumin-bilirubin.

3.2. ALBI score differs significantly among phases of chronic HBV infection in patients who are naïve to treatment

shown in Fig. 2A, ALBI score in HBeAg-positive CHB (−2.86 ± 0.39) or HBeAg-negative CHB (−2.96 ± 0.38) patients was significantly higher than that in HBeAg-positive chronic HBV infection −3.10 ± 0.26) or HBeAg-negative chronic HBV infection (−3.23 ± 0.24) (P < 0.001 between groups). Furthermore, patients were divided into two groups as grade 1 (≤−2.60) and grade 2

To gain insight on ALBI score throughout the natural history of chronic HBV infection, ALBI scores were analyzed among phases of chronic HBV infection in patients who were naïve to treatment. As

Fig. 1. Flowchart of the patient selection process. HBV, hepatitis B virus; HCV, hepatitis C virus; HBeAg, hepatitis B e antigen; PEG-IFN-α, pegylated interferon-alfa; NAs, nucleos(t)ide analogues. 122

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indices in different ALBI grade throughout the course of treatment were analyzed to investigate the influence of the baseline ALBI score on therapeutic effect in greater detail. Interestingly, the descent speed of HBsAg, HBV DNA, HBeAg and anti-HBe in patients with ALBI grade 2 was more rapid than that in patients with ALBI grade 1 during the course of treatment (Fig. 3E–H). However, no correlation was found between decreases in anti-HBc compared with the respective controls (Fig. 3I), both during the course of treatment (0–48 weeks) and at 24 weeks posttreatment. The above analyses indicate that HBeAg-positive CHB patients harbouring high baseline ALBI score exhibit a relatively stronger therapeutic response to PEG-IFN-α. 3.4. ALBI score correlates with the therapeutic response to NAs in HBeAgpositive CHB patients To identify whether there was a correlation between ALBI score and the anti-HBV therapeutic response to NAs, 122 HBeAg-positive CHB patients who had received anti-HBV treatment by NAs for 48 weeks were enrolled in this study. Baseline characteristics of the entire 122 HBeAg-positive CHB patients were shown in Supplementary Table 1. We compared the ALBI score between SR and NR groups who had received anti-HBV treatment by NAs. We found that the baseline ALBI score was higher in the SR group than in the NR group (−2.58 ± 0.45 versus −2.92 ± 0.32; P = 0.005) (Fig. 4A). Besides, the percentage of patients with ALBI grade 2 was higher in the SR group than that in the NR group (52.6% versus 17.5%; P < 0.001) (Fig. 4B). Furthermore, we analyzed the dynamic variations of ALBI score throughout the course of treatment. We found that only baseline ALBI score had significant differences between SR and NR groups (Fig. 4C). To investigate the influence of ALBI score on therapeutic response in greater detail, patients were divided into two groups according to baseline ALBI score. Among HBeAg-positive CHB patients, the SR rate of ALBI grade 2 was significantly higher than that of ALBI grade 1 after the follow-up of 72 weeks (35.7% versus 9.6%; P < 0.001, Fig. 4D). Then, the dynamic variations of six clinical serological and virological indices in different ALBI grade were analyzed throughout the course of treatment. We found that the descent speed of HBsAg, HBeAg and antiHBe in patients with ALBI grade 2 was more rapid than that in patients with ALBI grade 1 (Fig. 4E–G). However, no correlation was found between decreases in three indices (HBV DNA and anti-HBc) compared with the respective controls (Fig. 4H–I), both during the course of treatment and at 24 weeks posttreatment. Collectively, we conclude that HBeAg-positive CHB patients harbouring high baseline ALBI score exhibit a relatively stronger therapeutic response to NAs.

Fig. 2. ALBI score differs significantly across the natural history of chronic HBV infection. (A) Distribution of ALBI score throughout the natural progression of chronic HBV infection and healthy controls. (B–D) Association of ALBI grade with HBV markers in HBeAg-positive chronic hepatitis B patients. I, HBeAgpositive chronic infection; II, HBeAg-positive chronic hepatitis; III, BeAg-negative chronic infection; IV, HBeAg-negative chronic hepatitis; HC, healthy controls; ALBI, albumin-bilirubin; HBsAg, hepatitis B surface antigen; HBeAg, hepatitis B e antigen. Data are means ± SD. **P < 0.01, ***P < 0.001.

(> −2.60, ≤−1.39) according to ALBI score. As shown in Fig. 2B–D, patients with ALBI grade 1 have significantly higher serum HBsAg (P = 0.006) and HBeAg (P < 0.001) levels than patients with ALBI grade 2 in HBeAg-positive CHB patients. However, no differences in serum HBV DNA levels were observed between ALBI grade 1 and ALBI grade 2 group. Taken together, these data suggest that ALBI score was distributed differently among phases; specifically, ALBI score was highest in HBeAg-positive CHB patients and lowest in HBeAg-negative chronic HBV infection. Among HBeAg-positive CHB patients, ALBI grade was linked to the level of HBV markers.

3.3. ALBI score correlates with the therapeutic response to PEG-IFN-α in HBeAg-positive CHB patients To investigate whether there was a correlation between ALBI score and the anti-HBV therapeutic response to PEG-IFN-α, 121 HBeAg-positive CHB patients receiving PEG-IFN-α therapy for 48 weeks were enrolled in this study. Baseline characteristics of patients were shown in Supplementary Table 1. We compared the ALBI score between SR and NR groups who had received anti-HBV treatment by PEG-IFN-α. We found that the baseline ALBI score was higher in the SR group than in the NR group (−2.85 ± 0.32 versus −3.00 ± 0.28; P = 0.009) (Fig. 3A). Besides, the percentage of patients with ALBI grade 2 was higher in the SR group than that in the NR group (24.5% versus 8.8%; P < 0.001) (Fig. 3B). Furthermore, we analyzed the dynamic variations of ALBI score. We found that ALBI score was higher in the SR group than in the NR group throughout the course of treatment (Fig. 3C). To investigate the influence of ALBI score on therapeutic response in greater detail, patients were divided into two groups as ALBI grade 1 (≤−2.60) and ALBI grade 2 (> −2.60, ≤−1.39) according to baseline ALBI score. Among HBeAg-positive CHB patients, the SR rate of ALBI grade 2 was significantly higher than that of ALBI grade 1 after the follow-up of 72 weeks (68.4% versus 39.2%; P < 0.001, Fig. 3D). Then, the dynamic variations of six clinical serological and virological

3.5. ALBI score predicts SR to PEG-IFN-α or NAs therapy among HBeAgpositive CHB patients Logistic regression analysis was used to identify predictors of SR. For HBeAg-positive CHB patients treated with PEG-IFN-α, a univariate analysis model constructed in this study verified five protective effects of baseline serum TBIL (OR = 0.932, P = 0.039), DBIL (OR = 0.814, P = 0.013), ALT (OR = 0.996, P = 0.003), AST (OR = 0.994, P = 0.027) and ALBI score (OR = 0.19, P = 0.011) to achieve SR (Table 2). Conversely, baseline serum HBsAg (OR = 2.10, P = 0.040), HBeAg (OR = 6.84, P < 0.001) and anti-HBe (OR = 1.04, P < 0.001) were suggested as detrimental to SR achievement (Table 2). Multivariate logistic regression analysis that included the above significant variables identified eight parameters that influenced the SR. The baseline serum HBeAg was still suggested as detrimental to SR achievement (OR = 30.71, P = 0.008). Besides, ALT and ALBI score were still suggested as protective to SR achievement (OR = 0.994, P = 0.031; OR = 0.040, P = 0.003, respectively; Table 2). For HBeAgpositive CHB patients treated with NAs, univariable logistic regression 123

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Fig. 3. The baseline ALBI score correlates with the therapeutic response to PEG-IFN-α in HBeAg-positive CHB patients. (A and B) The baseline ALBI score and ALBI grade between the sustained response (SR) group (n = 53; left) and non-response (NR) group (n = 68; right) in HBeAg-positive CHB patients receiving PEG-IFNα therapy. (C) ALBI score from SR (blue) and NR (red) CHB patients at the indicated time points since PEG-IFN-α treatment. (D) The number of SR and NR patients between ALBI grade 1 group (n = 102; left) and ALBI grade 2 group (n = 19; right) in HBeAg-positive CHB patients. (E–I) The dynamic variations of clinical serological and virological indices (HBsAg, HBeAg, HBV DNA, anti-HBe and anti-HBc) in different ALBI grade throughout the course of PEG-IFN-α treatment. ALBI, albumin-bilirubin; PEG-IFN-α, pegylated interferon-alfa; HBeAg, hepatitis B e antigen; CHB, chronic hepatitis B; HBsAg, hepatitis B surface antigen; anti-HBe, hepatitis B e antibody; anti-HBc, hepatitis B core antibody. Data are means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

4. Discussion

analysis also identified seven parameters that influenced the SR. The baseline serum HBsAg (OR = 3.61, P = 0.037), HBeAg (OR = 8.67, P < 0.001) and anti-HBe (OR = 1.06, P < 0.001) were suggested as detrimental to SR achievement. In addition, we found four protective effects of baseline serum TBIL (OR = 0.929, P = 0.035), DBIL (OR = 0.856, P = 0.018), ALP (OR = 0.974, P = 0.004) and ALBI score (OR = 0.099, P = 0.001) to achieve SR (Table 2). However, no parameter appeared to correlate with therapy outcome by multivariate logistic regression analysis. To further evaluate the value for ALBI score to predict SR, we performed receiver operating characteristic (ROC) curve analysis. For HBeAg-positive CHB patients treated with PEG-IFN-α, the area under the curve (AUC) of ALBI score (0.649, 95% CI: 0.557–0.733) showed no significant difference in SR prediction when compared with ALT (0.645, 95% CI: 0.553–0.730), but was lower than that of log10 HBeAg (0.715, 95% CI: 0.626–0.794) (Fig. 5A). Interesting, the combined use of ALBI score, ALT and log10 HBeAg had higher AUC (0.807, 95% CI: 0.725–0.873) than single indicator (Fig. 5A). Meanwhile, for HBeAgpositive CHB patients treated with NAs, the AUC of ALBI score (0.705, 95% CI: 0.616–0.784) showed no significant difference in SR prediction when compared with log10 HBeAg (0.736, 95% CI: 0.645–0.815), but was higher than that of ALT (0.632, 95% CI: 0.539–0.718) (Fig. 5B). Interesting, the combined use of ALBI score, ALT and log10 HBeAg had higher AUC (0.860, 95% CI: 0.781–0.918) than single indicator (Fig. 5B). These results suggest that baseline ALBI score was statistical significantly correlated with an improved outcome in the logistic regression analysis, can be used as an effective predictor for identifying the response to PEG-IFN-α or NAs therapy before treatment for HBV infection.

The treatment timing choice and the clinical outcome prediction of CHB patients have attracted much attention of clinicians and researchers, which are important issues and difficult problems at present [22]. The current experimental diagnostic indicators are far from meeting the needs of accurate diagnosis of CHB. Finding new markers is the key to achieve an accurate diagnosis. Currently, clinical studies suggested that several factors were associated with PEG-IFN-α therapeutic efficacy in CHB patients [23–25]. However, the distribution of these indicators in each phase of CHB has not been explored. Additionally, some researches previously reported that serum HBV large surface protein [26] and HBV RNA [27–29] can be simultaneously used for distinguishing the different phases of HBV infection and predicting treatment response in CHB patients. Nevertheless, these indicators have not been commonly detected in clinic. The ideal indicator would be easy to detect before treatment, distinguish the different phases of chronic HBV infection, predict treatment response for CHB patients. Albumin-bilirubin involved only two variables of serum albumin and bilirubin, which could be easily acquired in clinic. Previous reports found that ALBI played a key role in predicting the long-term prognosis of HBV-related cirrhosis and HCC [19,21]. However, the roles of ALBI in regulating natural progression of chronic HBV infection and antiHBV treatment response are largely unknown. In this study, our findings clearly showed that ALBI score differed among phases of chronic HBV infection in patients who were naïve to treatment, with the highest score in HBeAg-positive CHB patients, followed by HBeAg-negative CHB patients, HBeAg-positive chronic HBV infection, and HBeAg-negative chronic HBV infection. This phenomenon may be caused by damaged liver function in active chronic hepatitis (HBeAg-positive and HBeAg-negative CHB) [9,30] which had 124

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Fig. 4. The baseline ALBI score correlates with the therapeutic response to NAs in HBeAg-positive CHB patients. (A and B) The baseline ALBI score and ALBI grade between the sustained response (SR) group (n = 19; left) and non-response (NR) group (n = 103; right) in HBeAgpositive CHB patients receiving NAs therapy. (C) ALBI score from SR (blue) and NR (red) CHB patients at the indicated time points since NAs treatment. (D) The number of SR and NR patients between ALBI grade 1 group (n = 94; left) and ALBI grade 2 group (n = 28; right) in HBeAg-positive CHB patients. (E–I) The dynamic variations of clinical serological and virological indices (HBsAg, HBeAg, HBV DNA, anti-HBe and anti-HBc) in different ALBI grade throughout the course of NAs treatment. ALBI, albumin-bilirubin; NAs, nucleos(t)ide analogues; HBeAg, hepatitis B e antigen; CHB, chronic hepatitis B; HBsAg, hepatitis B surface antigen; anti-HBe, hepatitis B e antibody; anti-HBc, hepatitis B core antibody. Data are means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 2 Logistic regression analysis to predict achieving sustained response to PEG-IFN-α or NAs therapy in HBeAg-positive CHB patients. Baseline variables

PEG-IFN-α treatment

NAs treatment

Univariate analysis

Gender Age BMI, kg/m2 Log10 HBV DNA, IU/ml Log10 HBsAg, IU/ml Log10 HBeAg, S/CO Anti-HBe, S/CO Anti-HBc, S/CO Total bilirubin, μmol/L Direct bilirubin, μmol/L Indirect bilirubin, μmol/L Total protein, g/L Albumin, g/L Globin, g/L ALT, U/L AST, U/L ALT/AST GGT, U/L LDH, U/L ALP, U/L Cholinesterase, U/L ALBI score

Multivariate analysis

Univariate analysis

Multivariate analysis

ORa (95% CI)

P value

OR (95% CI)

P value

ORa (95% CI)

P value

ORa (95% CI)

P value

2.18 (0.95–4.99) 1.04 (0.97–1.12) 1.08 (0.94–1.28) 1.50 (0.97–2.33) 2.10 (1.04–4.27) 6.84 (2.32–20.10) 1.04 (1.02–1.06) 0.98 (0.89–1.08) 0.932 (0.872–0.997) 0.814 (0.693–0.957) 0.927 (0.838–1.026) 1.02 (0.96–1.10) 1.11 (0.99–1.24) 0.98 (0.89–1.07) 0.996 (0.993–0.999) 0.994 (0.988–0.999) 0.813 (0.53–1.26) 0.998 (0.990–1.005) 0.998 (0.992–1.004) 0.994 (0.978–1.010) 1.00 (1.00–1.00) 0.19 (0.05–0.67)

0.066 0.284 0.361 0.069 0.040 < 0.001 < 0.001 0.716 0.039 0.013 0.145 0.503 0.062 0.619 0.003 0.027 0.352 0.513 0.511 0.436 0.432 0.011

– – – – 0.58 (0.18–1.80) 30.71 (2.40–393.20) 1.00 (0.97–1.04) – 0.86 (0.68–1.09) 1.73 (0.93–3.20) – – – – 0.994 (0.988–0.999) 1.00 (0.99–1.01) – – – – – 0.040 (0.005–0.345)

– – – – 0.341 0.008 0.806 – 0.203 0.082 – – – – 0.031 0.892 – – – – – 0.003

1.18 (0.38–3.60) 1.00 (0.92–1.09) 1.38 (0.94–1.88) 1.48 (0.81–2.71) 3.61 (1.08–12.04) 8.67 (2.97–25.32) 1.06 (1.03–1.10) 1.21 (0.96–1.51) 0.929 (0.868–0.995) 0.856 (0.753–0.973) 0.96 (0.85–1.08) 1.05 (0.97–1.15) 1.20 (0.95–1.36) 0.93 (0.81–1.06) 0.998 (0.994–1.001) 0.995 (0.990–1.000) 1.13 (0.60–2.12) 0.995 (0.987–1.003) 0.992 (0.981–1.004) 0.974 (0.956–0.992) 1.00 (1.00–1.00) 0.099 (0.026–0.383)

0.777 0.997 0.185 0.200 0.037 < 0.001 < 0.001 0.107 0.035 0.018 0.491 0.222 0.066 0.267 0.162 0.068 0.713 0.245 0.199 0.004 0.064 0.001

– – – – 0.82 (0.14–4.88) 3.23 (0.21–50.67) 1.05 (0.97–1.14) – 0.96 (0.66–1.38) 1.00 (0.53–1.90) – – – – – – – – – 0.995 (0.966–1.026) – 0.176 (0.016–1.896)

– – – – 0.828 0.404 0.264 – 0.811 1.000 – – – – – – – – – 0.766 – 0.152

Bold font indicates statistical significance (P < 0.05). a Odds ratio (OR) > 1 indicates a detrimental association with response to treatment. OR < 1 indicates a protective association with response to treatment. PEGIFN-α, pegylated interferon-alfa; NAs, nucleos(t)ide analogues; HBeAg, hepatitis B e antigen; CHB, chronic hepatitis B; CI, confidence interval; BMI, body mass index; HBsAg, hepatitis B surface antigen; anti-HBe, hepatitis B e antibody; anti-HBc, hepatitis B core antibody; ALT, alanine aminotransferase; AST, aspartate amino transferase; GGT, gamma-glutamyltransferase; LDH, lactic dehydrogenase; ALP, alkaline phosphatase; ALBI, albumin-bilirubin. 125

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Fig. 5. The combined use of ALBI score and other markers enhances the predictive value of treatment efficiency in HBeAg-positive CHB patients. (A) ROC curves of ALT, log10 HBeAg and ALBI score in predicting sustained response (SR) before PEG-IFN-α treatment. (B) ROC curves of ALT, log10 HBeAg and ALBI score in predicting SR before NAs treatment.ALBI, albumin-bilirubin; PEG-IFN-α, pegylated interferon-alfa; NAs, nucleos(t)ide analogues; HBeAg, hepatitis B virus e antigen; CHB, chronic hepatitis B; ALT, alanine aminotransferase; AUC, area under the curve; ROC, receiver operating characteristic curve.

baseline ALBI score can be used as a clinical predictor for predicting therapy response before clinical PEG-IFN-α or NAs treatment. Fourthly, the combined use of ALBI score, HBeAg and ALT may be a potential model for predicting SR. Certain limitations of our study should be also mentioned. Firstly, the ALBI score of only 19 SR patients was examined in HBeAg-positive CHB patients who received NAs therapy because of very low HBeAg seroconversion rate [22], which may have reduced our statistical power. Secondly, there were no ALBI grade 3 patients in the cohort because they were mostly diagnosed with decompensated liver cirrhosis or liver cancer. Thirdly, the mechanisms underlying regulation of ALBI to viral replication during different phases of CHB and treatment response during the course of treatment will be needed for further investigation. Fourthly, a certain proportion of CHB patients were assigned to receive PEG-IFN-α add-on NAs. To investigate whether the treatment regimen affects the ALBI score, we compared the ALBI score from CHB patients treated with either PEG-IFN-α monotherapy or PEGIFN-α/NAs add-on at the indicated time points since PEG-IFN-α treatment. However, no significant differences were detected at any time point (data not shown). Additionally, to better understand the initial chosen of anti-viral drugs, we compared ALBI score between PEG-IFN-α and NAs groups. However, no significant differences were detected at any time point since PEG-IFN-α or NAs treatment (data not shown). In conclusion, our findings identify a previously unrecognized role of ALBI score in each phase of HBV natural history and HBeAg-positive CHB patients during PEG-IFN-α or NAs treatment. ALBI score differs significantly across the natural course of chronic HBV infection and ALBI score is correlated with PEG-IFN-α or NAs therapeutic response in HBeAg-positive CHB patients. Moreover, these findings present an auxiliary clinical indicator for predicting stronger efficacy of PEG-IFN-α or NAs therapy in CHB patients. Hence, monitoring of ALBI score may help clinicians in managing chronic HBV infection, aiding in decisions on the initiation of therapy, predicting of treatment response for individuals during clinical practice.

elevated total bilirubin levels and constant albumin levels as shown in Table 1. Thus, ALBI score provided an important reference for initial evaluation and follow-up assessments in guiding antiviral treatment during clinical practice. Furthermore, we explored the ALBI score in HBeAg-positive CHB patients treated with PEG-IFN-α or NAs to investigate the association of ALBI score with long-term outcome under treatment. The present study demonstrated that a majority of SR CHB patients harbored high baseline ALBI score and ALBI grade compared to NR CHB patients whether receiving PEG-IFN-α or NAs therapy. Interestingly, CHB patients possessing high ALBI grade showed the higher HBeAg and HBsAg loss rate in response to anti-HBV therapy throughout the course of treatment. Besides, the dynamic analysis also showed that the descent speed of HBsAg, HBV DNA, HBeAg and anti-HBe in patients with ALBI grade 2 was more rapid than that in patients with ALBI grade 1 throughout the course of treatment. These results revealed that CHB patients harbouring high ALBI grade exhibited a relatively stronger therapeutic response to PEG-IFN-α or NAs. What’s more, the univariate regression analysis suggested that high ALT concentrations, low HBsAg concentrations and low HBeAg concentrations at baseline associated with SR to PEG-IFN-α or NAs, which were consistent with previous reports [31–33]. In current study, the ALBI score, log10 HBeAg and ALT were both independent factors for treatment response of PEG-IFN-α. Especially, ALT is an accepted indictor for treatment response of PEG-IFN-α. According to the analysis of AUCs of them, ALBI score showed a better ability than ALT in predicting SR. Interesting, the combined use of ALBI score, HBeAg and ALT could enhance the efficiency of predicting SR to PEG-IFN-α or NAs treatment compared with single HBeAg or ALT. So far, there have not commonly used predicting models. The combined use of ALBI score, HBeAg and ALT may be a potential model for predicting SR. The above analysis indicated that high ALBI score was associated with SR to achieve durable HBeAg seroconversion induced by PEG-IFN-α therapy or NAs. ALBI contributed to the response to antiHBV therapy in CHB patients and might be useful for predicting response to treatment. Considered together, ALBI score may potentially be used for clinical implementation based on our observations. Firstly, it is easy to be detected in clinic. Secondly, ALBI score demonstrates significant differences across the different phases of chronic HBV infection. Thirdly,

Author contributions All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. 126

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Ethical approval [11]

The study was approved by the Institutional Medical Ethics Review Board of the First Affiliated Hospital of Fujian Medical University (Grant No. FY2013017).

[12]

CRediT authorship contribution statement [13]

Zhen Xun: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft, Writing - review & editing. Can Liu: Methodology, Resources, Data curation, Funding acquisition. Qing-Qing Yu: Validation, Investigation. Jin-Piao Lin: Conceptualization, Supervision. Jin-Lan Huang: Validation. Ting-Wen Yang: Investigation. Wen-Nan Wu: Formal analysis, Visualization. Song-Hang Wu: Investigation, Data curation. Qi-Shui Ou: Conceptualization, Resources, Writing - original draft, Supervision, Project administration, Funding acquisition.

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[15]

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Declaration of Competing Interest The authors declare no conflicts of interest.

[17]

Acknowledgements [18]

The authors are grateful to the staff and patients of our clinics for the provision of the samples used in this study. This work was supported by the National Natural Science Foundation of China [grant number 81971996, 81672101, 81902130, 81871710] and the Startup Fund for scientific research of Fujian Medical University [grant number 2018QH2038].

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Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cca.2019.12.020.

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