Chronic viral B hepatitis (CHB) is a potentially life-threatening liver disease that may progress to liver failure and cirrhosis. Currently, although combinations of different laboratory methods are used in the follow-up and treatment of CHB, the failure of these procedures in some cases has led to the necessity of developing new approaches. In CHB, the intrahepatic expression pattern of viral antigens, including hepatitis B surface antigen (HBsAg), is related to different phases of inflammation. However, many studies have focused on the intracytoplasmic properties of HBsAg staining, and HBsAg positivity in liver tissue has not been evaluated by objective quantitative methods.

To investigate the relationship of image analysis-based quantitative HBsAg expression and its staining patterns with clinicopathological factors and treatment in CHB.

A total of 140 liver biopsies from treatment-naïve cases with CHB infection were included in this study. Following diagnosis, all patients were treated with entecavir (0.5 mg) and followed up at three-month intervals. The percentage of immunohistochemical HBsAg (p-HBsAg) expression in the liver was determined in whole tissue sections of biopsies from each case by image analysis. The immunohistochemical staining pattern was also evaluated separately according to 3 different previously defined classifications.

A positive correlation between p-HBsAg and serum levels of hepatitis B virus (HBV) DNA and HBsAg was observed (P < 0.001). The p-HBsAg value was significantly higher in younger patients than in older patients. When the groups were categorized according to the hepatitis B e antigen (HBeAg) status in HBeAg-positive cases, p-HBsAg was correlated with HBV DNA, hepatitis activity index (HAI) and fibrosis scores (P < 0.001). In this group, p-HBsAg and HBsAg expression patterns were also correlated with the viral response (VR) and the serological response (SR) (P < 0.001). Multivariate analysis revealed that p-HBsAg was an independent predictor of either VR or SR (P < 0.001). In HBeAg-negative patients, although HBsAg expression patterns were correlated with both HAI and fibrosis, no relationship was observed among p-HBsAg, clinicopathological factors and VR.

In pretreatment liver biopsies, the immunohistochemical determination of HBsAg expression by quantitative methods, beyond its distribution within the cell, may be a good predictor of the treatment response, especially in HBeAg-positive cases.

Despite advances in its prevention, diagnosis and treatment, chronic viral B hepatitis (CHB) continues to be an important worldwide health problem with high morbidity and mortality due to its dramatic consequences, such as cirrhosis and hepatocellular carcinoma[1]. In recent years, researchers have suggested that the combination of various laboratory methods with histopathological findings may allow the assessment of the prognosis and treatment of patients with CHB[2-4]. High liver biopsy activity scores, low viremia and high serum alanine aminotransferase (ALT) levels are associated with hepatitis B e antigen (HBeAg) seroconversion, while a decrease in serum hepatitis B surface antigen (HBsAg) levels is among the factors associated with an effective response to therapy[5-7]. Unfortunately, the presence of these factors is not always adequate to determine the progression of the disease and the efficacy of treatment. The treatment response is not sufficient in some patients with these factors, which contrasts with the adequate response in other patients without these factors, suggesting that other viral and host-related parameters may also be involved in the progression and therapy response.

Recent studies have also provided evidence that in patients with CHB, the expression of viral antigens and their expression patterns detected by immunohistochemistry may be related to clinicopathological factors and might have prognostic implications for the disease process. The expression of hepatitis B core antigen (HBcAg) and its expression patterns were found to be associated with serum HBeAg, viral replication and the hepatitis activity index (HAI)[8-11]. In addition, the results of some studies have suggested the potential use of HBcAg expression as a marker of treatment response[12-14].

Regarding HBsAg, the results of many different studies investigating the relationships between the intrahepatic expression of this antigen revealed that differences in the intracytoplasmic HBsAg staining pattern were related to viremia and active inflammation[14-18]. In these investigations, HBsAg expression was evaluated semiquantitatively, and different classifications were used for the HBsAg staining pattern. On the other hand, HBsAg, beyond being the main diagnostic marker of the disease, is a very important tool in the follow-up of the course and treatment of CHB, since a decrease in its level reflects the low replicative phase[19-21]. Therefore, more objective methods are needed to determine whether the immunohistochemical expression of HBsAg in pretreatment-performed liver biopsies can be useful in monitoring the prognosis and treatment of CHB.

Therefore, this study was undertaken to investigate the relationship between image analysis-based quantitation of HBsAg expression and corresponding staining patterns with clinicopathological factors and treatment.

The mean age of the patients in the whole group was 42.79 ± 11.53 years, and the male to female ratio was 1.25 (Table 1). There was a positive correlation between p-HBsAg and the serum levels of HBV DNA and HBsAg (r: 0.490, r: 0.468 P < 0.001, respectively). When all cases were grouped according to mean age, the p-HBsAg value was significantly higher in younger patients than in older patients (48.47 ± 16.06 vs 31.72 ± 12.89 P < 0.01). According to their HBeAg status, 86 cases were HBeAg positive (61.5%), and 54 cases were HBeAg negative (38.5%). Other findings of the cases are presented in Table 1. Briefly, HBeAg-positive patients were younger than HBeAg-negative patients, and serum HBV DNA and HBsAg levels were higher in this group (P < 0.05). On the other hand, the INR levels were found to be lower than those in the HBeAg-negative group. The average p-HBsAg level was significantly higher in the HBeAg-negative group than in the HBeAg-positive group. There was no difference in the HBsAg staining patterns between the two groups.

The relationship between viremia and histopathological findings in cases with positive HBeAg are summarized in Table 2. The average p-HBsAg level was found to be significantly higher in patients with higher HBV DNA values than in those with low HBV DNA levels. In contrast, an inverse relationship was noted between p-HBsAg and either HAI or the fibrosis score. Regarding staining pattern 1 (SP1), the number of cases with pattern A was higher among cases with higher HBV DNA levels. Their number decreased among cases with HAI ≥ 9, and it was not observed among cases with fibrosis. When staining pattern 2 (SP2) was analyzed, it was noted that the number of cases with diffuse staining was higher in the HAI ≥ 9 group. In contrast, among the cases with high HAI and fibrosis scores, the frequency of pattern E was observed in 42% and 75% of the cases, respectively.

The relationship between viremia and histopathological findings in cases with negative HBeAg is presented in Table 3. In this group, no correlation was observed between either the p-HBsAg or the membranous staining pattern and HBV DNA, HAI, or fibrosis. In the evaluation of patients according to SP1, in cases with HAI ≥ 9, the number of patients with pattern A was lower than that of patients with HAI < 9. In the latter, pattern E was rarely observed (12% of cases). SP2 was found to be related to HBV DNA levels, and diffuse staining was more frequent in cases with a higher level of HBV DNA. However, globular staining was rarely observed in these cases.

Patients were treated for a median duration of 28 mo (range, 4 mo to 62 mo). VR was achieved in 54 of 86 HBeAg-positive patients (66.7%) and in all 54 HBeAg-negative patients (100%), with a median time to VR of 16 and 9 mo, respectively. Factors associated with VR are shown in Table 4. In HBeAg-positive patients, the p-HBsAg and HBsAg expression patterns were correlated with VR, along with age, fibrosis, HVDNA and HBsAg levels in the log-rank test (P < 0.05). VR was observed in 53.7% of the patients with a high p-HBsAg. Similarly, pattern A and the presence of membranous expression were frequently observed in patients with VR. In multivariate analysis, the sole independent factor associated with VR was p-HBsAg (P < 0.01)(Figure 2A and Table 5). Forty-seven (54.6%) of 86 HBeAg-positive patients exhibited HBeAg SR, with a median time of 22 mo (range, 4 mo to 60 mo). Univariate analysis revealed that the p-HBsAg and HBsAg expression patterns were correlated with SR together with HBsAg levels and ALT > 200 (P < 0.05) (Table 4). Multivariate analysis demonstrated that p-HBsAg and the HAI score were independent factors related to SR (Figure 2B and C and Table 5). On the other hand, in the HBeAg-negative group, the most valuable determinant of VR was found to be the HAI score (Figure 2D and Table 5).

Open in New Tab   Full Size Figure   Download Figure

Figure 2 Kaplan-Meier analysis in hepatitis B surface antigen positive and hepatitis B e antigen negative cases (P < 0.01). A: Cumulative incidence of viral response in hepatitis B e antigen (HBeAg) positive patients is more frequently observed in cases with higher percentage of immunohistochemical hepatitis B surface antigen (p-HBsAg) expression; B-D: In the same group, serological response is also significantly related to p-HBsAg expression (B) and HBsAg levels (C). However, in HBeAg negative in patients, VR is closely related to hepatitis activity index (D). p-HBsAg: Percentage of immunohistochemical hepatitis B surface antigen; HBsAg: Hepatitis B surface antigen; HAI: Hepatitis activity index; VR: Viral response; SR: Serological response.

The few previous studies that evaluated HBsAg expression by semiquantitative methods have shown that serum HBsAg levels are correlated with the number of intrahepatic HBsAg-positive cells[19,20]. This observation was interpreted as serum and hepatic HBsAg being interrelated. On the other hand, in our study, we determined the percentage of intrahepatic HBsAg expression in the whole tissue section of each case by image analysis. Our results showed that in HBeAg-positive cases, p-HBsAg was associated not only with serum HBsAg but also with serum HBV DNA levels, indicating that intrahepatic HBsAg in liver tissues is closely related to viremia. Recently, Su et al[25] investigated HBsAg expression in the liver and its relationship with both serum HbsAg and interferon therapy using morphometric methods in treatment-naïve HbeAg-positive patients. Parallel to the findings of the present study, they observed a strong correlation between hepatic HbsAg expression and serum HbsAg levels. In addition, the relationship between HbsAg level and HbeAg loss after interferon therapy observed in this study suggested that the HbsAg expression level may be a useful marker to determine the response to therapy.

Therefore, in our study, we also aimed to elucidate the relationship between p-HbsAg levels and the treatment response in patients receiving entecavir treatment together with other HbsAg expression patterns. In the HbeAg-positive group, VR and SR were observed more frequently in cases with pretreatment values of p-HbsAg levels above the group average. In the HbeAg-positive group, the pretreatment values of p-HbsAg were strongly related to either VR or SR, suggesting the clinical significance of p-HbsAg in the management of HbeAg-positive CHB patients with antiviral treatment. Moreover, multivariate analysis revealed that in this group, p-HbsAg was an independent factor associated with VR and SR, warranting further investigation to evaluate the potential role of p-HbsAg as a tool to predict the response to therapy.

Another interesting result of this study is the presence of an inverse relationship between p-HbsAg and HAI and fibrosis scores in the HbeAg-positive group. Parallel to this finding, in cases with high HAI and/or fibrosis scores, the serum HBV DNA and HbsAg levels were significantly lower, suggesting that in the liver, the replacement of hepatocytes by both increased inflammatory infiltration and increased connective tissue can lead to this situation.

Our study demonstrated that HbsAg expression patterns, evaluated by various immunohistochemical methods, have different relationships with clinicopathological parameters and treatment responses in patients with CHB. Many years ago, following the detection of ground-glass hepatocytes by immunohistochemical methods in the liver tissues of patients with anti-Hbe positivity in the serum and a low HBV DNA level, the relationship of HbsAg expression patterns in hepatocytes with clinicopathological factors was investigated.Some studies have shown that patterns A and B, as opposed to those of C and D, are more common in younger subjects[15,18]. However, no similar age-related difference finding in HbsAg staining patterns was observed in other studies[16]. In our study, most of the patients with patterns A and B were younger than those with patterns C and D.

When the relationship of these staining patterns with disease activity and viremia has been investigated in recent studies, most of the data indicate that patterns A and B are associated with viremia[15,18,21,26,27]. In parallel with this finding, in our study, these patterns were associated with high HBV DNA and HAI in the HbeAg-positive group and were rarely observed in cases with high fibrosis scores, supporting the hypothesis that both are associated with active disease. Interestingly, the frequent detection of pattern B in the HbeAg-negative group, especially in cases with high HAI scores, also points to a relationship with active disease in these cases[15]. While the preliminary results indicate that the marginal staining pattern (pattern E) may be a determinant of inactive virus replication, other studies concluded that pattern C may be a marker of this situation[15,28]. In our cases, pattern E was more frequent in cases with low levels of HBV DNA and more frequent in cases with high fibrosis scores in the HbeAg-positive group, supporting data about its relationship with inactive disease reported by previous studies. Similar to recent findings in HbeAg-positive cases, membranous expression was absent in 84% and 87% of patients with lower HBV DNA values and fibrosis scores, respectively[18]. These findings suggest that this pattern of staining reflects active liver disease.

In an elegant study, Yim et al[26] investigated the relationship between these staining patterns and antiviral treatment response in patients with CHB. Diffuse staining patterns were associated with SR in HBeAg-positive patients. However, multivariate analysis revealed that this was not an independent predictor. In our study, although HBsAg expression patterns were associated with VR and SR in univariate analysis, they were not found to be independent factors. In contrast, p-HBsAg was a good predictor of the response to therapy in HBeAg-positive cases. This finding emphasizes that the determination of HBsAg expression by quantitative methods, beyond its distribution within the cell, may be an objective marker that can be used during the course and treatment of the disease.

In recent years, there has been growing interest in developing various noninvasive methods for monitoring the course and treatment of CHB. On the other hand, although it is an expensive and invasive method, liver biopsy remains the gold standard in determining liver injury and fibrosis during the follow-up of the disease. However, beyond these parameters, our results emphasize that the HBsAg expression levels measured in pretreatment liver biopsies via quantitative methods may be a useful marker for determining the treatment response.

This study demonstrated that HBsAg expression patterns, evaluated by various immunohistochemical methods, have different relationships with clinicopathological parameters and the treatment response in patients with CHB. Although the expression pattern of HBsAg differs significantly according to the disease status, it shows lower clinical significance in predicting the response to therapy than the percentage of HBsAg expression. Therefore, the quantitative evaluation of HBsAg expression in pretreatment-naïve liver biopsies of patients with CHB may provide a prediction of the response to antiviral therapy, especially in cases with HBeAg positivity. However, our findings should be supported by additional large studies.