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Predictive factors for risk of hepatocellular carcinoma in immune inactive chronic hepatitis B
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Clinics and Research in Hepatology and Gastroenterology (2020) xxx, xxx—xxx
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ORIGINAL ARTICLE
Predictive factors for risk of hepatocellular carcinoma in immune inactive chronic hepatitis B Seung In Seo , Hyoung Su Kim ∗, Bo Kyung Yang , Jin Gu Kang , Woon Geon Shin , Jin Heon Lee , Hak Yang Kim , Myoung Kuk Jang Department of Internal Medicine, Kangdong Sacred Heart Hospital, Hallym University College of Medicine, 150, Sungnae-gil, Kangdong-gu, Seoul, Republic of Korea 134-701
KEYWORDS Immune inactive phase; Chronic hepatitis B; Hepatocellular carcinoma
∗
Summary Background and aims: The risk factors for hepatocellular carcinoma (HCC) in immune inactive chronic hepatitis B (CHB) have not been clarified. The aim of this study was to investigate the predictive factors for HCC inimmune inactive CHB. Methods: A total of 337 patients in immune inactive CHB were consecutively enrolled in Kangdong Sacred Heart Hospital from 1995 to 2017. Univariate and multivariate analyses were performed to identify the independent risk factors for HCC development. Results: During the mean 63 months of follow-up, the incidence of HCC of study population was 4.5% (15/337). Patients who developed HCC were older, had more cirrhosis at baseline, and were more likely to experience ALT elevation > 2 X upper limit of normal (ULN) during follow-up than those without HCC. In Cox regression analysis, increased ALT levels > 2 X ULN during follow-up (hazard ratio [HR], 3.774; 95% confidence interval [CI], 1.145—12.443; P = 0.029] and presence of cirrhosis (HR, 11.768; 95% CI, 3.350—41.336; P < 0.001) were identified as the independent factors for HCC in immune inactive CHB. With increasing number of risk factors, the respective cumulative incidence of HCC at 10 years was 6.3%, 8.8%, and 63.5%. Conclusions: Underlying cirrhosis and hepatic inflammation reflected by increased ALT levels > 2 X ULN were significant predictors for HCC in immune inactive CHB. ALT elevation showed a synergistic effect in HCC development combined with cirrhosis. It suggests that patients with high serum ALT levels, especially those with cirrhosis, are required closer surveillance for HCC even in immune inactive CHB. © 2020 Elsevier Masson SAS. All rights reserved.
Corresponding author. E-mail address: [email protected] (H.S. Kim).
https://doi.org/10.1016/j.clinre.2019.10.009 2210-7401/© 2020 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Seo SI, et al. Predictive factors for risk of hepatocellular carcinoma in immune inactive chronic hepatitis B. Clin Res Hepatol Gastroenterol (2020), https://doi.org/10.1016/j.clinre.2019.10.009
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Introduction Chronic hepatitis B virus (HBV) infection is a worldwide public health challenge and one of the major causes of liver-related morbidity and mortality [1,2]. The clinical spectrum of HBV infection ranges from simple chronic hepatitis through cirrhosis to eventual hepatocellular carcinoma (HCC). The natural course of chronic hepatitis B (CHB) is highly variable among patients, some of whom remain in immune inactive CHB, whereas others develop active hepatitis or cirrhosis [2]. Generally, immune inactive CHB, defined as patients with chronic HBV infection characterized as hepatitis B envelope antigen (HBeAg) negative, antibody to HBeAg (anti-HBe) positive, who have HBV DNA levels undetectable or lower than 2000 IU/mL, along with persistently normal serum alanine aminotransferase (ALT) levels, have a good long-term prognosis and are not recommended for antiviral therapy [3]. Regarding HCC epidemiology, the majority of HBV-related HCCs develop in cirrhotic livers, but approximately 20% develop in non-cirrhotic livers [2]. The risk factors for HCC in patients with CHB can be classified into host, viral, and environmental factors. Factors include older age, male sex, family history of HCC, cirrhosis, HBV genotype, HBV mutant, high level of HBV DNA, elevated ALT levels, alcohol, smoking, metabolic syndrome, and other concurrent viral infection such as hepatitis C virus (HCV), hepatitis D virus (HDV), and human immunodeficiency virus (HIV) [4]. The REVEAL-HBV cohort studies have shown that serum HBV DNA levels and ALT levels are predominant drivers of disease progression in patients with CHB [5,6]. Although the immune inactive CHB rarely progresses to advanced diseases such as cirrhosis or HCC, recent studies have suggested that some inactive HBV carriers have a substantial risk of HCC and liver-related death [7—9]. To date, the risk factors for HCC in patients with CHB have been evaluated in all phases of CHB, including active or inactive phases. Therefore, it has to be elucidated whether known risk factors for HCC in CHB could affect progression in immune inactive CHB. The present study aimed to identify the risk factors for HCC development in immune inactive CHB based on various clinical parameters including the patterns of long-term change in serum ALT or HBV DNA levels.
Materials and methods Patients and study design A total of 337 patients in immune inactive CHB were consecutively enrolled in Kangdong Sacred Heart Hospital from 1995 to 2017. The follow-up duration was calculated from the initial visit to the last visit or the time of diagnosis of HCC. Patients were recruited to the study as immune inactive CHB if they met the following criteria: • age ≥ 18 years with chronic HBV infection defined by the presence of hepatitis B surface antigen (HBsAg) for more than 6 months;
• both HBeAg negative and anti-HBe positive; • persistently normal serum ALT levels within 1 year of baseline(upper limit of normal [ULN] ≤ 40 IU/L); • serum HBV DNA levels at baseline < 2000 IU/mL; • no previous history of any antiviral therapies. Exclusion criteria were as follows: • decompensated cirrhosis showing evidence of significant portal hypertension indicated by the presence of ascites, esophageal or gastric varices, and/or hepatic encephalopathy; • chronic alcohol consumption > 20 g/d or history of use of drugs known to cause hepatitis; • concomitant HCV, HDV, or HIV infection at time of enrollment; • follow-up duration less than 1 year; • any other malignancies; • HCC diagnosed within 1 year of enrollment. All patients underwent follow-up at regular intervals of every 3—6 months. Demographic data such as age at initial visit, sex, and other clinical information were obtained by review of the medical records. Biochemistry, serum tumor markers, including alpha-fetoprotein, and serology for HBV, including serum HBV-DNA levels, were routinely evaluated at every visit. Imaging studies such as ultrasonography or computed tomography were performed for HCC surveillance every 6 to 12 months. HCC was diagnosed either histologically or clinically based on the guidelines of the American Association for the Study of Liver Diseases [10]. Baseline characteristics were compared between the non-HCC and HCC groups on the basis of various clinical parameters. Univariate and multivariate analyses were performed to identify the independent risk factors associated with the development of HCC. The study was approved by the Institutional Review Board of the study institution and the protocol conforms to the ethical guidelines of the Declaration of Helsinki.
Serologic testing Testing was performed for serologic markers for HBV, including HBsAg, antibody to HBsAg (anti-HBs), HBeAg, and anti-HBe, using commercial assay kits (Abbott Laboratories, Chicago, IL, USA). Serum HBV DNA was quantified using the bDNA assay (VersantTM 3.0, lower limit of detection, 2000 copies/ml; Bayer HealthCare LLC Diagnostic Division, Tarrytown, NY, USA) until December 2008 or COBAS TaqMan PCR assay (lower limit of detection, 116 copies/mL; Roche Diagnostics, Branchburg, NJ, USA) after January 2009.
Statistical analysis Baseline characteristics were compared with the MannWhitney U test or chi-squared test, as appropriate. The cumulative incidence of HCC was analyzed using the KaplanMeier method, and differences were determined with the log-rank test. Univariate and multivariate analyses were performed to identify the independent risk factors associated with the development of HCC using Cox proportional
Please cite this article in press as: Seo SI, et al. Predictive factors for risk of hepatocellular carcinoma in immune inactive chronic hepatitis B. Clin Res Hepatol Gastroenterol (2020), https://doi.org/10.1016/j.clinre.2019.10.009
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HCC in immune inactive CHB Table 1
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Baseline patient characteristics.
Age, years Men, n (%) Baseline DNA (IU/mL) ALT (IU/L) Bilirubin (mg/dL) Albumin (mg/dL) Platelets (K) Cirrhosis, n (%) During follow-up HBV DNA elevation (> 2000IU/mL), n (%) Antiviral therapy, n (%) ALT elevation > ULN, n (%) AL Televation > 2xULN, n (%) HBsAg loss, n (%)
Total (n = 337)
Non-HCC(n = 322)
HCC (n = 15)
P value
50 (18—87) 186 (55.2) 192 (20—1844) 21 (4—40) 0.7 (0.2—3.3) 4.4 (2.1—-5.3) 212 (38—511) 43 (12.8)
56 (21—94) 176 (54.7) 400 (20—1939) 22 (3—40) 0.7 (0.2—3.9) 4.4 (2.3—5.3) 215 (25—511) 32 (9.9)
62 (42—75) 10 (66.7) 400 (20—1248) 25 (15—31) 0.9 (0.2—2.2) 3.7 (3.0—4.9) 201 (94—284) 11 (73.3)
0.035 0.433 0.936 0.097 0.668 0.279 0.051 < 0.001
58 (17.2) 12 (3.6) 115 (34.1) 40 (11.9) 55 (16.3)
55 (17.1) 9 (2.8) 104 (32.3) 31 (9.6) 52 (16.1)
3 (20.0) 3 (20.0) 11 (73.3) 9 (60.0) 3 (20.0)
0.729 0.012 0.002 < 0.001 0.719
ALT: alanine aminotransferase; ULN, upper limit of normal; HBsAg: hepatitis B surface antigen; HBV: hepatitis B virus; HCC: hepatocellular carcinoma. Data are expressed as median (range) or number.
Figure 1
Natural course of immune inactive CHB. A. Cumulative rates of HBsAg loss. B. Cumulative rates of HCC development.
hazards regression models. A P value less than 0.05 was considered statistically significant. All statistical analyses were carried out using SPSS for Windows, Version 24.0 (IBM Corp., Armonk, NY, USA).
Results Baseline characteristics and natural course The baseline characteristics of the study population are summarized in Table 1. One hundred eighty-six (55.2%) patients were men and the mean age was 48.6 ± 12.8 years. A history of cirrhosis was present in 43 (12.8%) patients. The mean HBV DNA levels and ALT levels were 309 ± 364 IU/mL and 22.6 ± 9.0 IU/L, respectively. Major route of transmission was vertical transmission. The mean follow-up duration was 63 ± 43 months. There were no patients with co-infection of HCV, HDV or HIV during the follow-up period. HBsAg loss was observed in 55 patients. Cumulative rates of HBsAg loss at 1, 2, 3, 5, 7, 9, 11, and 13 years were 1.2%, 3.3%, 5.4%, 12.4%, 24.5%, 26.6%, 30.2%, and 58.1%, respectively, during the follow-up period (Fig. 1A). The loss gradually increased with the duration of follow-up. During the follow-up period, HBV DNA levels were elevated above 2000 IU/mL in 17.2% (58 of 337) of patients but most of these elevations were transient and not associated
with ALT elevation. Only 3.6% of all patients (12 of 337) started antiviral therapies. During the follow-up period, 34.1% of the entire study population experienced ALT elevation > ULN (40 IU/L).
Predictive factors for HCC risk among all patients During follow-up, HCC was newly diagnosed in 15 patients (4.5%). The cumulative rates of HCC development at 1, 2, 3, 5, 7, 9, 11, and 13 years were 0.6%, 1.7%, 3.3%, 4.9%, 5.7%, 7.0%, 10.6%, and 10.6%, respectively, during the follow-up period (Fig. 1B). Of the baseline clinical features, older age and cirrhosis (11/15 [73.3%] vs. 32/322 [9.9%], P < 0.001) were considered risk factors for HCC. Other factors such as sex, baseline DNA levels, and baseline ALT levels were not significantly different between the non-HCC group and the HCC group. During the follow-up period, elevation of HBV DNA levels above 2000 IU/mL was observed in 17.2% (58 of 337) of patients, but the proportions were not significantly different between the two groups (Table 1). During follow-up, 34.1% of all study subjects experienced ALT elevation > ULN (40 IU/L). There were more patients in the HCC group than in the non-HCC group with increased ALT levels (73.3% [11/15] vs. 32.3% [104/322], P = 0.002). Similarly, there were more patients with increased ALT levels > 2 X ULN during follow-up in the HCC group (60.0% [9/15] vs. 9.6% [31/322], P < 0.001).
Please cite this article in press as: Seo SI, et al. Predictive factors for risk of hepatocellular carcinoma in immune inactive chronic hepatitis B. Clin Res Hepatol Gastroenterol (2020), https://doi.org/10.1016/j.clinre.2019.10.009
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S.I. Seo et al. Table 2
Univariate and multivariate analysis of risk factors for predicting HCC development. Univariate analysis
Age Cirrhosis Antiviral therapy ALTelevation > 2xULN DNA elevation > 2000 IU/mL
Multivariate analysis
HR
95% CI
P value
HR
95% CI
P value
1.053 18.236 4.714 8.956 1.038
1.006—1.102 5.801—57.327 1.324—16.792 3.177—25.247 0.293—3.682
0.025 < 0.001 0.017 < 0.001 0.954
1.053 11.768 1.412 3.774 1.148
0.989—1.121 3.350—41.336 0.257—7.765 1.145—12.443 0.261—5.048
0.103 < 0.001 0.692 0.029 0.855
ALT: alanine aminotransferase; ULN: upper limit of normal; HCC: hepatocellular carcinoma; HR: hazard ratio; CI: confidence interval.
Table 3
HCC risk according to risk factors.
Cirrhosis/ALT elevation > 2xULN No cirrhosis/No ALT elevation Cirrhosis/No ALT elevation No cirrhosis/ALT elevation Cirrhosis/ALT elevation
N
HCC, n
HR
270 27 24 16
3 3 1 8
Referent 9.003 2.616 44.062
95% CI
P value < 0.001
1.794—45.180 0.270—25.324 11.503—168.771
0.008 0.406 < 0.001
ALT: alanine aminotransferase; ULN: upper limit of normal; HCC: hepatocellular carcinoma; HR: hazard ratio; CI: confidence interval.
Cox regression analysis was used to evaluate factors associated with the development of HCC, which included age, cirrhosis, antiviral therapy, HBV DNA elevation (> 2000 IU/mL), and increased ALT levels > 2 X ULN. Taken together, cirrhosis (hazard ratio [HR], 11.768; 95% confidence interval [CI], 3.350—41.336; P < 0.001] and increased ALT levels > 2 XULN (HR, 3.774; 95% CI, 1.145—12.443; P = 0.029] were found to be independent factors associated with HCC development (Table 2). HBV DNA elevation during follow-up was not associated with HCC development in inactive HBV carriers. The combined effects of cirrhosis and ALT elevation showed a statistically significant and synergistic association with HCC risk, with the highest risk observed in patients with both cirrhosis and ALT elevation (HR, 44.062; 95% CI, 11.503—168.771; P < 0.001; Table 3). After exclusion of 16 patients with both risk factors, only cirrhosis was associated with HCC development, but increased ALT levels were not.
rates of HCC development at 1, 3, 5, 7, 9, 11,and 13years were 0.7%, 1.1%, 1.1%, 1.1%, 1.1%, 5.6%, and 5.6% in patients without cirrhosis and 2.6%, 16.7%, 27.8%, 33.0%, 39.1%, 39.1%, and 39.1% in patients with cirrhosis, respectively (P < 0.001; Fig. 2A). Cumulative rates of HCC development at 1, 3, 5, 7, 9, 11,and 13years were 0.3%, 2.1%, 2.1%, 2.1%, 2.1%, 6.5%, and 6.5% in patients without ALT elevation and 2.5%, 10.8%, 20.1%, 24.1%, 29.1%, 29.1%, and 29.1% in patients with ALT elevation, respectively (P < 0.001; Fig. 2B). HCC occurred in only 1.1% (3/270) of patients without risk factors (no cirrhosis and no ALT elevation) and in 7.8% (4/51) of patients with one risk factor. However, patients with two risk factors were diagnosed with HCC in 50% of cases (8/16). During the observation period, the respective cumulative incidence of HCC at 10 years according to increasing number of favorable predictors was 6.3%, 8.8%, and 63.5%. There was a significant difference among the groups (P < 0.001; Fig. 3).
Impact of risk factors for HCC
Discussion
Patients with cirrhosis or ALT elevation > 2 X ULN had a significantly higher probability of HCC development. Cumulative
In the present retrospective longitudinal cohort study, we confirmed that the risk of HCC development was still con-
Figure 2 HCC development according to presence of risk factors. A. Cumulative rates of HCC development according to cirrhosis (P < 0.001). B. Cumulative rates of HCC development according to ALT elevation (P < 0.001).
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HCC in immune inactive CHB
Figure 3 HCC development according to number of risk factors. Cumulative rates of HCC development in patients with 2, 1, and 0 risk factors are presented (P < 0.001).
siderable in patients inimmune inactive CHB. During the follow-up period, the cumulative incidence of HCC at 13 years, 10.6%, was not as low as we speculated. In multivariate analysis, in addition to the presence of cirrhosis, increased ALT levels > 2 X ULN were found to be an independent risk factor for HCC development. Many previous epidemiologic studies have demonstrated the risk factors for HCC in patients with CHB, which can be classified into host, viral, and environmental factors, such as older age, male sex, family history of HCC, cirrhosis, HBV genotype, HBV mutant, high level of HBV DNA, elevated ALT levels, alcohol, smoking, metabolic syndrome, and other concurrent viral infection. Among them, cirrhosis has been considered as a most important risk factor for HCC. In our study, baseline cirrhosis was present in 43 patients (12.8%). HCC was newly developed in 11 patients of cirrhotic patients (11/43) but only 4 patients in non-cirrhotic patients (4/337). Recently, serial changes in serum HBV DNA and ALT levels were evaluated as risk factors and considered to be predominant drivers of disease progression in patients with CHB [4—6,9]. A few studies in the past have performed long-term follow-up of immune inactive CHB. These studies have shown that immune inactive CHB were still at risk for HCC [7—9]. A long-term large-scale study reporting the predictors of liver disease progression in immune inactive CHB [7] included only subjects in non-cirrhotic immune inactive CHB who were compared with individuals not infected with HBV, with a 13year follow-up period. According to the study, the annual incidence of HCC was higher in immune inactive CHB than in the control group (0.06% vs. 0.02%). Moreover, older age and alcohol consumption were independent predictors for HCC in multivariate analysis. However, the study did not consider dynamic changes of HBV DNA and ALT levels during the follow-up period. Tai and colleagues also conducted a longterm follow-up study with non-cirrhotic immune inactive CHB during a mean follow-up of 13.4 years [9]. In the study, age, sex, and maximal ALT levels were significant independent factors associated with the development of cirrhosis, HCC, and mortality [9]. However, the study focused only on ALT levels during follow-up, without considering HBV DNA levels. Another recent study documented the importance of HBV DNA levels for HCC development even in compensated cirrhotic patients with low viral loads [8]. That is, the risk
5 of HCC in patients with detectable but low viral loads during follow-up was significantly higher than that in those with undetectable HBV DNA levels. Because this study was conducted in patients with cirrhosis only, the results might not be generalizable to all immune inactive CHB. In that point, the present study is meaningful that it documented increased ALT levels as a predictor for HCC development after adjusting for serial HBV DNA levels and other risk factors including cirrhosis in immune inactive CHB. Serum ALT levels are reportedly the most common serologic marker of HBV-related liver cell injury or hepatic inflammation [11,12]. All guidelines regarding CHB treatment, therefore, recommend that patients with active HBV replication and serum ALT levels > 2 X ULN are candidates for antiviral therapy [13—16]. In real-practice, higher ALT levels imply a more active immune response against HBV and more extensive hepatocyte damage. Subsequently, chronic inflammation associated with human immune responses to HBV infection may increase liver damage, resulting in proliferation of hepatocytes. Finally, persistent injury and regeneration may promote liver carcinogenesis [5,17]. Persistent necro inflammation may accelerate the progression to cirrhosis and the development of HCC in CHB [18]. Several prospective studies have shown that elevated serum ALT levels at enrollment were a risk factor for HBV-related HCC [6,19]. Recent studies evaluating liver histology in immune inactive CHB with persistently normal alanine aminotransferase (PNALT) [20—22] suggested that the PNALT levels were associated with milder fibrosis or inflammation. Considering these findings, the ALT assay, as it is less expensive than concentrating HBV DNA levels and can be performed easily, may be used as a good ancillary tool for HCC surveillance in immune inactive CHB at short intervals during long-term follow up. While serum HBV DNA levels were considered a major factor for progression of liver disease in patients with CHB, baseline and serial HBV DNA changes were not associated with HCC development in the present study. This result might be caused by the relatively small number of patients experiencing reactivation and the control of HBV DNA by antiviral agent, if indicated. An interesting finding of our study was that cirrhosis and increased ALT levels showed a synergistic association with HCC risk. The HRs for HCC of cirrhosis and increased ALT levels were 9.003 and 2.616, respectively. However, when both factors were combined, the HR for HCC increased to 44.062. Serum HBsAg could be used to predict intrahepatic covalently closed circular DNA (cccDNA) and HBV DNA level in CHB patients with low serum HBeAg statues, especially in HBeAg negative patients [23], although, recently, serum hepatitis B core-related antigen is known to be a more satisfactory factor for surrogate marker for cccDNA [24]. Clinically, there is increased recognition that loss of HBsAg is the most reliable indicator to measure cure of HBV infection. Previous longitudinal studies indicated that the annual rate of HBsAg loss is approximately 0.5—2.3%, depending on the age and status of liver disease at enrollment [25—27]. In the present study, we demonstrated that in immune inactive CHB, the cumulative incidence rate of HBsAg loss at 10 years was 30.2%. The higher probability of HBsAg loss in our study might be the result of baseline characteristics such as older age and immune inactive CHB. HCC developed in only three among 55 patients who experienced HBsAg loss, and
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all three had cirrhosis before HBsAg loss. That is, the prognosis after HBsAg loss was good as long as cirrhosis did not emerge before HBsAg loss, supporting the finding that HBsAg loss confers a favorable clinical outcome or even the cure of disease. There are some limitations in the present study. It should be noted that almost all Korean patients with CHB are infected with HBV genotype C [28], which is known to progress more rapidly to HCC than other genotypes. Therefore, it could be confounding factors and it would be necessary to analyze other genotypes in the future. Our study did not evaluate other risk factors such as HBV mutants and metabolic syndrome. Lastly, our study was a retrospective study, conducted in a single center and with a relatively small number of patients. These findings should be further validated in larger studies with longer follow-up periods. In conclusion, in addition to the presence of cirrhosis, a high degree of hepatic inflammation reflected by increased ALT levels > 2 X ULN during the follow-up period was also a significant predictor for HCC in immune inactive CHB. ALT elevation showed a synergistic effect in HCC development in combination with cirrhosis. These findings suggest that patients who have high serum ALT levels, especially those with cirrhosis, require meticulous surveillance for HCC even in immune inactive CHB.
Disclosure of interest The authors declare that they have no competing interest.
Financial support None.
Author contributions Seung In Seo: study concept and design, data acquisition, data analysis, drafting of the manuscript, statistical analysis. Hyoung Su Kim: study concept and design, data analysis, drafting of the manuscript, study supervision. Jin Gu Kang: statistical analysis. Woon Geon Shin: administrative support. Jin Heon Lee: data acquisition. Hak Yang Kim: critical revision of the manuscript. Myoung Kuk Jang: study concept and design, study supervision, revision of the manuscript.
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ORIGINAL ARTICLE
Predictive factors for risk of hepatocellular carcinoma in immune inactive chronic hepatitis B Seung In Seo , Hyoung Su Kim ∗, Bo Kyung Yang , Jin Gu Kang , Woon Geon Shin , Jin Heon Lee , Hak Yang Kim , Myoung Kuk Jang Department of Internal Medicine, Kangdong Sacred Heart Hospital, Hallym University College of Medicine, 150, Sungnae-gil, Kangdong-gu, Seoul, Republic of Korea 134-701
KEYWORDS Immune inactive phase; Chronic hepatitis B; Hepatocellular carcinoma
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Summary Background and aims: The risk factors for hepatocellular carcinoma (HCC) in immune inactive chronic hepatitis B (CHB) have not been clarified. The aim of this study was to investigate the predictive factors for HCC inimmune inactive CHB. Methods: A total of 337 patients in immune inactive CHB were consecutively enrolled in Kangdong Sacred Heart Hospital from 1995 to 2017. Univariate and multivariate analyses were performed to identify the independent risk factors for HCC development. Results: During the mean 63 months of follow-up, the incidence of HCC of study population was 4.5% (15/337). Patients who developed HCC were older, had more cirrhosis at baseline, and were more likely to experience ALT elevation > 2 X upper limit of normal (ULN) during follow-up than those without HCC. In Cox regression analysis, increased ALT levels > 2 X ULN during follow-up (hazard ratio [HR], 3.774; 95% confidence interval [CI], 1.145—12.443; P = 0.029] and presence of cirrhosis (HR, 11.768; 95% CI, 3.350—41.336; P < 0.001) were identified as the independent factors for HCC in immune inactive CHB. With increasing number of risk factors, the respective cumulative incidence of HCC at 10 years was 6.3%, 8.8%, and 63.5%. Conclusions: Underlying cirrhosis and hepatic inflammation reflected by increased ALT levels > 2 X ULN were significant predictors for HCC in immune inactive CHB. ALT elevation showed a synergistic effect in HCC development combined with cirrhosis. It suggests that patients with high serum ALT levels, especially those with cirrhosis, are required closer surveillance for HCC even in immune inactive CHB. © 2020 Elsevier Masson SAS. All rights reserved.
Corresponding author. E-mail address: [email protected] (H.S. Kim).
https://doi.org/10.1016/j.clinre.2019.10.009 2210-7401/© 2020 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Seo SI, et al. Predictive factors for risk of hepatocellular carcinoma in immune inactive chronic hepatitis B. Clin Res Hepatol Gastroenterol (2020), https://doi.org/10.1016/j.clinre.2019.10.009
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Introduction Chronic hepatitis B virus (HBV) infection is a worldwide public health challenge and one of the major causes of liver-related morbidity and mortality [1,2]. The clinical spectrum of HBV infection ranges from simple chronic hepatitis through cirrhosis to eventual hepatocellular carcinoma (HCC). The natural course of chronic hepatitis B (CHB) is highly variable among patients, some of whom remain in immune inactive CHB, whereas others develop active hepatitis or cirrhosis [2]. Generally, immune inactive CHB, defined as patients with chronic HBV infection characterized as hepatitis B envelope antigen (HBeAg) negative, antibody to HBeAg (anti-HBe) positive, who have HBV DNA levels undetectable or lower than 2000 IU/mL, along with persistently normal serum alanine aminotransferase (ALT) levels, have a good long-term prognosis and are not recommended for antiviral therapy [3]. Regarding HCC epidemiology, the majority of HBV-related HCCs develop in cirrhotic livers, but approximately 20% develop in non-cirrhotic livers [2]. The risk factors for HCC in patients with CHB can be classified into host, viral, and environmental factors. Factors include older age, male sex, family history of HCC, cirrhosis, HBV genotype, HBV mutant, high level of HBV DNA, elevated ALT levels, alcohol, smoking, metabolic syndrome, and other concurrent viral infection such as hepatitis C virus (HCV), hepatitis D virus (HDV), and human immunodeficiency virus (HIV) [4]. The REVEAL-HBV cohort studies have shown that serum HBV DNA levels and ALT levels are predominant drivers of disease progression in patients with CHB [5,6]. Although the immune inactive CHB rarely progresses to advanced diseases such as cirrhosis or HCC, recent studies have suggested that some inactive HBV carriers have a substantial risk of HCC and liver-related death [7—9]. To date, the risk factors for HCC in patients with CHB have been evaluated in all phases of CHB, including active or inactive phases. Therefore, it has to be elucidated whether known risk factors for HCC in CHB could affect progression in immune inactive CHB. The present study aimed to identify the risk factors for HCC development in immune inactive CHB based on various clinical parameters including the patterns of long-term change in serum ALT or HBV DNA levels.
Materials and methods Patients and study design A total of 337 patients in immune inactive CHB were consecutively enrolled in Kangdong Sacred Heart Hospital from 1995 to 2017. The follow-up duration was calculated from the initial visit to the last visit or the time of diagnosis of HCC. Patients were recruited to the study as immune inactive CHB if they met the following criteria: • age ≥ 18 years with chronic HBV infection defined by the presence of hepatitis B surface antigen (HBsAg) for more than 6 months;
• both HBeAg negative and anti-HBe positive; • persistently normal serum ALT levels within 1 year of baseline(upper limit of normal [ULN] ≤ 40 IU/L); • serum HBV DNA levels at baseline < 2000 IU/mL; • no previous history of any antiviral therapies. Exclusion criteria were as follows: • decompensated cirrhosis showing evidence of significant portal hypertension indicated by the presence of ascites, esophageal or gastric varices, and/or hepatic encephalopathy; • chronic alcohol consumption > 20 g/d or history of use of drugs known to cause hepatitis; • concomitant HCV, HDV, or HIV infection at time of enrollment; • follow-up duration less than 1 year; • any other malignancies; • HCC diagnosed within 1 year of enrollment. All patients underwent follow-up at regular intervals of every 3—6 months. Demographic data such as age at initial visit, sex, and other clinical information were obtained by review of the medical records. Biochemistry, serum tumor markers, including alpha-fetoprotein, and serology for HBV, including serum HBV-DNA levels, were routinely evaluated at every visit. Imaging studies such as ultrasonography or computed tomography were performed for HCC surveillance every 6 to 12 months. HCC was diagnosed either histologically or clinically based on the guidelines of the American Association for the Study of Liver Diseases [10]. Baseline characteristics were compared between the non-HCC and HCC groups on the basis of various clinical parameters. Univariate and multivariate analyses were performed to identify the independent risk factors associated with the development of HCC. The study was approved by the Institutional Review Board of the study institution and the protocol conforms to the ethical guidelines of the Declaration of Helsinki.
Serologic testing Testing was performed for serologic markers for HBV, including HBsAg, antibody to HBsAg (anti-HBs), HBeAg, and anti-HBe, using commercial assay kits (Abbott Laboratories, Chicago, IL, USA). Serum HBV DNA was quantified using the bDNA assay (VersantTM 3.0, lower limit of detection, 2000 copies/ml; Bayer HealthCare LLC Diagnostic Division, Tarrytown, NY, USA) until December 2008 or COBAS TaqMan PCR assay (lower limit of detection, 116 copies/mL; Roche Diagnostics, Branchburg, NJ, USA) after January 2009.
Statistical analysis Baseline characteristics were compared with the MannWhitney U test or chi-squared test, as appropriate. The cumulative incidence of HCC was analyzed using the KaplanMeier method, and differences were determined with the log-rank test. Univariate and multivariate analyses were performed to identify the independent risk factors associated with the development of HCC using Cox proportional
Please cite this article in press as: Seo SI, et al. Predictive factors for risk of hepatocellular carcinoma in immune inactive chronic hepatitis B. Clin Res Hepatol Gastroenterol (2020), https://doi.org/10.1016/j.clinre.2019.10.009
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HCC in immune inactive CHB Table 1
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Baseline patient characteristics.
Age, years Men, n (%) Baseline DNA (IU/mL) ALT (IU/L) Bilirubin (mg/dL) Albumin (mg/dL) Platelets (K) Cirrhosis, n (%) During follow-up HBV DNA elevation (> 2000IU/mL), n (%) Antiviral therapy, n (%) ALT elevation > ULN, n (%) AL Televation > 2xULN, n (%) HBsAg loss, n (%)
Total (n = 337)
Non-HCC(n = 322)
HCC (n = 15)
P value
50 (18—87) 186 (55.2) 192 (20—1844) 21 (4—40) 0.7 (0.2—3.3) 4.4 (2.1—-5.3) 212 (38—511) 43 (12.8)
56 (21—94) 176 (54.7) 400 (20—1939) 22 (3—40) 0.7 (0.2—3.9) 4.4 (2.3—5.3) 215 (25—511) 32 (9.9)
62 (42—75) 10 (66.7) 400 (20—1248) 25 (15—31) 0.9 (0.2—2.2) 3.7 (3.0—4.9) 201 (94—284) 11 (73.3)
0.035 0.433 0.936 0.097 0.668 0.279 0.051 < 0.001
58 (17.2) 12 (3.6) 115 (34.1) 40 (11.9) 55 (16.3)
55 (17.1) 9 (2.8) 104 (32.3) 31 (9.6) 52 (16.1)
3 (20.0) 3 (20.0) 11 (73.3) 9 (60.0) 3 (20.0)
0.729 0.012 0.002 < 0.001 0.719
ALT: alanine aminotransferase; ULN, upper limit of normal; HBsAg: hepatitis B surface antigen; HBV: hepatitis B virus; HCC: hepatocellular carcinoma. Data are expressed as median (range) or number.
Figure 1
Natural course of immune inactive CHB. A. Cumulative rates of HBsAg loss. B. Cumulative rates of HCC development.
hazards regression models. A P value less than 0.05 was considered statistically significant. All statistical analyses were carried out using SPSS for Windows, Version 24.0 (IBM Corp., Armonk, NY, USA).
Results Baseline characteristics and natural course The baseline characteristics of the study population are summarized in Table 1. One hundred eighty-six (55.2%) patients were men and the mean age was 48.6 ± 12.8 years. A history of cirrhosis was present in 43 (12.8%) patients. The mean HBV DNA levels and ALT levels were 309 ± 364 IU/mL and 22.6 ± 9.0 IU/L, respectively. Major route of transmission was vertical transmission. The mean follow-up duration was 63 ± 43 months. There were no patients with co-infection of HCV, HDV or HIV during the follow-up period. HBsAg loss was observed in 55 patients. Cumulative rates of HBsAg loss at 1, 2, 3, 5, 7, 9, 11, and 13 years were 1.2%, 3.3%, 5.4%, 12.4%, 24.5%, 26.6%, 30.2%, and 58.1%, respectively, during the follow-up period (Fig. 1A). The loss gradually increased with the duration of follow-up. During the follow-up period, HBV DNA levels were elevated above 2000 IU/mL in 17.2% (58 of 337) of patients but most of these elevations were transient and not associated
with ALT elevation. Only 3.6% of all patients (12 of 337) started antiviral therapies. During the follow-up period, 34.1% of the entire study population experienced ALT elevation > ULN (40 IU/L).
Predictive factors for HCC risk among all patients During follow-up, HCC was newly diagnosed in 15 patients (4.5%). The cumulative rates of HCC development at 1, 2, 3, 5, 7, 9, 11, and 13 years were 0.6%, 1.7%, 3.3%, 4.9%, 5.7%, 7.0%, 10.6%, and 10.6%, respectively, during the follow-up period (Fig. 1B). Of the baseline clinical features, older age and cirrhosis (11/15 [73.3%] vs. 32/322 [9.9%], P < 0.001) were considered risk factors for HCC. Other factors such as sex, baseline DNA levels, and baseline ALT levels were not significantly different between the non-HCC group and the HCC group. During the follow-up period, elevation of HBV DNA levels above 2000 IU/mL was observed in 17.2% (58 of 337) of patients, but the proportions were not significantly different between the two groups (Table 1). During follow-up, 34.1% of all study subjects experienced ALT elevation > ULN (40 IU/L). There were more patients in the HCC group than in the non-HCC group with increased ALT levels (73.3% [11/15] vs. 32.3% [104/322], P = 0.002). Similarly, there were more patients with increased ALT levels > 2 X ULN during follow-up in the HCC group (60.0% [9/15] vs. 9.6% [31/322], P < 0.001).
Please cite this article in press as: Seo SI, et al. Predictive factors for risk of hepatocellular carcinoma in immune inactive chronic hepatitis B. Clin Res Hepatol Gastroenterol (2020), https://doi.org/10.1016/j.clinre.2019.10.009
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S.I. Seo et al. Table 2
Univariate and multivariate analysis of risk factors for predicting HCC development. Univariate analysis
Age Cirrhosis Antiviral therapy ALTelevation > 2xULN DNA elevation > 2000 IU/mL
Multivariate analysis
HR
95% CI
P value
HR
95% CI
P value
1.053 18.236 4.714 8.956 1.038
1.006—1.102 5.801—57.327 1.324—16.792 3.177—25.247 0.293—3.682
0.025 < 0.001 0.017 < 0.001 0.954
1.053 11.768 1.412 3.774 1.148
0.989—1.121 3.350—41.336 0.257—7.765 1.145—12.443 0.261—5.048
0.103 < 0.001 0.692 0.029 0.855
ALT: alanine aminotransferase; ULN: upper limit of normal; HCC: hepatocellular carcinoma; HR: hazard ratio; CI: confidence interval.
Table 3
HCC risk according to risk factors.
Cirrhosis/ALT elevation > 2xULN No cirrhosis/No ALT elevation Cirrhosis/No ALT elevation No cirrhosis/ALT elevation Cirrhosis/ALT elevation
N
HCC, n
HR
270 27 24 16
3 3 1 8
Referent 9.003 2.616 44.062
95% CI
P value < 0.001
1.794—45.180 0.270—25.324 11.503—168.771
0.008 0.406 < 0.001
ALT: alanine aminotransferase; ULN: upper limit of normal; HCC: hepatocellular carcinoma; HR: hazard ratio; CI: confidence interval.
Cox regression analysis was used to evaluate factors associated with the development of HCC, which included age, cirrhosis, antiviral therapy, HBV DNA elevation (> 2000 IU/mL), and increased ALT levels > 2 X ULN. Taken together, cirrhosis (hazard ratio [HR], 11.768; 95% confidence interval [CI], 3.350—41.336; P < 0.001] and increased ALT levels > 2 XULN (HR, 3.774; 95% CI, 1.145—12.443; P = 0.029] were found to be independent factors associated with HCC development (Table 2). HBV DNA elevation during follow-up was not associated with HCC development in inactive HBV carriers. The combined effects of cirrhosis and ALT elevation showed a statistically significant and synergistic association with HCC risk, with the highest risk observed in patients with both cirrhosis and ALT elevation (HR, 44.062; 95% CI, 11.503—168.771; P < 0.001; Table 3). After exclusion of 16 patients with both risk factors, only cirrhosis was associated with HCC development, but increased ALT levels were not.
rates of HCC development at 1, 3, 5, 7, 9, 11,and 13years were 0.7%, 1.1%, 1.1%, 1.1%, 1.1%, 5.6%, and 5.6% in patients without cirrhosis and 2.6%, 16.7%, 27.8%, 33.0%, 39.1%, 39.1%, and 39.1% in patients with cirrhosis, respectively (P < 0.001; Fig. 2A). Cumulative rates of HCC development at 1, 3, 5, 7, 9, 11,and 13years were 0.3%, 2.1%, 2.1%, 2.1%, 2.1%, 6.5%, and 6.5% in patients without ALT elevation and 2.5%, 10.8%, 20.1%, 24.1%, 29.1%, 29.1%, and 29.1% in patients with ALT elevation, respectively (P < 0.001; Fig. 2B). HCC occurred in only 1.1% (3/270) of patients without risk factors (no cirrhosis and no ALT elevation) and in 7.8% (4/51) of patients with one risk factor. However, patients with two risk factors were diagnosed with HCC in 50% of cases (8/16). During the observation period, the respective cumulative incidence of HCC at 10 years according to increasing number of favorable predictors was 6.3%, 8.8%, and 63.5%. There was a significant difference among the groups (P < 0.001; Fig. 3).
Impact of risk factors for HCC
Discussion
Patients with cirrhosis or ALT elevation > 2 X ULN had a significantly higher probability of HCC development. Cumulative
In the present retrospective longitudinal cohort study, we confirmed that the risk of HCC development was still con-
Figure 2 HCC development according to presence of risk factors. A. Cumulative rates of HCC development according to cirrhosis (P < 0.001). B. Cumulative rates of HCC development according to ALT elevation (P < 0.001).
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HCC in immune inactive CHB
Figure 3 HCC development according to number of risk factors. Cumulative rates of HCC development in patients with 2, 1, and 0 risk factors are presented (P < 0.001).
siderable in patients inimmune inactive CHB. During the follow-up period, the cumulative incidence of HCC at 13 years, 10.6%, was not as low as we speculated. In multivariate analysis, in addition to the presence of cirrhosis, increased ALT levels > 2 X ULN were found to be an independent risk factor for HCC development. Many previous epidemiologic studies have demonstrated the risk factors for HCC in patients with CHB, which can be classified into host, viral, and environmental factors, such as older age, male sex, family history of HCC, cirrhosis, HBV genotype, HBV mutant, high level of HBV DNA, elevated ALT levels, alcohol, smoking, metabolic syndrome, and other concurrent viral infection. Among them, cirrhosis has been considered as a most important risk factor for HCC. In our study, baseline cirrhosis was present in 43 patients (12.8%). HCC was newly developed in 11 patients of cirrhotic patients (11/43) but only 4 patients in non-cirrhotic patients (4/337). Recently, serial changes in serum HBV DNA and ALT levels were evaluated as risk factors and considered to be predominant drivers of disease progression in patients with CHB [4—6,9]. A few studies in the past have performed long-term follow-up of immune inactive CHB. These studies have shown that immune inactive CHB were still at risk for HCC [7—9]. A long-term large-scale study reporting the predictors of liver disease progression in immune inactive CHB [7] included only subjects in non-cirrhotic immune inactive CHB who were compared with individuals not infected with HBV, with a 13year follow-up period. According to the study, the annual incidence of HCC was higher in immune inactive CHB than in the control group (0.06% vs. 0.02%). Moreover, older age and alcohol consumption were independent predictors for HCC in multivariate analysis. However, the study did not consider dynamic changes of HBV DNA and ALT levels during the follow-up period. Tai and colleagues also conducted a longterm follow-up study with non-cirrhotic immune inactive CHB during a mean follow-up of 13.4 years [9]. In the study, age, sex, and maximal ALT levels were significant independent factors associated with the development of cirrhosis, HCC, and mortality [9]. However, the study focused only on ALT levels during follow-up, without considering HBV DNA levels. Another recent study documented the importance of HBV DNA levels for HCC development even in compensated cirrhotic patients with low viral loads [8]. That is, the risk
5 of HCC in patients with detectable but low viral loads during follow-up was significantly higher than that in those with undetectable HBV DNA levels. Because this study was conducted in patients with cirrhosis only, the results might not be generalizable to all immune inactive CHB. In that point, the present study is meaningful that it documented increased ALT levels as a predictor for HCC development after adjusting for serial HBV DNA levels and other risk factors including cirrhosis in immune inactive CHB. Serum ALT levels are reportedly the most common serologic marker of HBV-related liver cell injury or hepatic inflammation [11,12]. All guidelines regarding CHB treatment, therefore, recommend that patients with active HBV replication and serum ALT levels > 2 X ULN are candidates for antiviral therapy [13—16]. In real-practice, higher ALT levels imply a more active immune response against HBV and more extensive hepatocyte damage. Subsequently, chronic inflammation associated with human immune responses to HBV infection may increase liver damage, resulting in proliferation of hepatocytes. Finally, persistent injury and regeneration may promote liver carcinogenesis [5,17]. Persistent necro inflammation may accelerate the progression to cirrhosis and the development of HCC in CHB [18]. Several prospective studies have shown that elevated serum ALT levels at enrollment were a risk factor for HBV-related HCC [6,19]. Recent studies evaluating liver histology in immune inactive CHB with persistently normal alanine aminotransferase (PNALT) [20—22] suggested that the PNALT levels were associated with milder fibrosis or inflammation. Considering these findings, the ALT assay, as it is less expensive than concentrating HBV DNA levels and can be performed easily, may be used as a good ancillary tool for HCC surveillance in immune inactive CHB at short intervals during long-term follow up. While serum HBV DNA levels were considered a major factor for progression of liver disease in patients with CHB, baseline and serial HBV DNA changes were not associated with HCC development in the present study. This result might be caused by the relatively small number of patients experiencing reactivation and the control of HBV DNA by antiviral agent, if indicated. An interesting finding of our study was that cirrhosis and increased ALT levels showed a synergistic association with HCC risk. The HRs for HCC of cirrhosis and increased ALT levels were 9.003 and 2.616, respectively. However, when both factors were combined, the HR for HCC increased to 44.062. Serum HBsAg could be used to predict intrahepatic covalently closed circular DNA (cccDNA) and HBV DNA level in CHB patients with low serum HBeAg statues, especially in HBeAg negative patients [23], although, recently, serum hepatitis B core-related antigen is known to be a more satisfactory factor for surrogate marker for cccDNA [24]. Clinically, there is increased recognition that loss of HBsAg is the most reliable indicator to measure cure of HBV infection. Previous longitudinal studies indicated that the annual rate of HBsAg loss is approximately 0.5—2.3%, depending on the age and status of liver disease at enrollment [25—27]. In the present study, we demonstrated that in immune inactive CHB, the cumulative incidence rate of HBsAg loss at 10 years was 30.2%. The higher probability of HBsAg loss in our study might be the result of baseline characteristics such as older age and immune inactive CHB. HCC developed in only three among 55 patients who experienced HBsAg loss, and
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all three had cirrhosis before HBsAg loss. That is, the prognosis after HBsAg loss was good as long as cirrhosis did not emerge before HBsAg loss, supporting the finding that HBsAg loss confers a favorable clinical outcome or even the cure of disease. There are some limitations in the present study. It should be noted that almost all Korean patients with CHB are infected with HBV genotype C [28], which is known to progress more rapidly to HCC than other genotypes. Therefore, it could be confounding factors and it would be necessary to analyze other genotypes in the future. Our study did not evaluate other risk factors such as HBV mutants and metabolic syndrome. Lastly, our study was a retrospective study, conducted in a single center and with a relatively small number of patients. These findings should be further validated in larger studies with longer follow-up periods. In conclusion, in addition to the presence of cirrhosis, a high degree of hepatic inflammation reflected by increased ALT levels > 2 X ULN during the follow-up period was also a significant predictor for HCC in immune inactive CHB. ALT elevation showed a synergistic effect in HCC development in combination with cirrhosis. These findings suggest that patients who have high serum ALT levels, especially those with cirrhosis, require meticulous surveillance for HCC even in immune inactive CHB.
Disclosure of interest The authors declare that they have no competing interest.
Financial support None.
Author contributions Seung In Seo: study concept and design, data acquisition, data analysis, drafting of the manuscript, statistical analysis. Hyoung Su Kim: study concept and design, data analysis, drafting of the manuscript, study supervision. Jin Gu Kang: statistical analysis. Woon Geon Shin: administrative support. Jin Heon Lee: data acquisition. Hak Yang Kim: critical revision of the manuscript. Myoung Kuk Jang: study concept and design, study supervision, revision of the manuscript.
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