Hepatitis B Virus Infection is a Prognostic Biomarker for Better Survival in Operable Esophageal Cancer: Analysis of 2,004 Patients from an Endemic Area in China Free

Esophageal cancer is one of the most common and aggressive cancers in China, with more than 470,000 new cases and 370,000 deaths annually (1). Despite improvement of surgical techniques and incorporation of new therapeutic approaches for decades, esophageal cancer is still a highly devastating disease with poor prognosis, 5-year overall survival at less than 40% (2, 3). The TNM stage system is currently the most reliable method to predict the outcome of patients. However, patients even with the same TNM stage might have dramatically different prognosis (4). Thus, the identification of new prognostic factors is of great importance to improve outcome prediction and tailor individualized treatment in patients with esophageal cancer.

China has a high prevalence of hepatitis B virus (HBV) infection, with hepatitis B surface antigen (HBsAg) seropositivity rates between 10% to 12% in the general population (5). Chronic HBV infection is the most common cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma (6). Besides, HBV is identified to be associated with other cancers, such as lymphoma (7), leukemia (8), and pancreatic cancer (9). Previous studied indicated that HBV infection had an influence on the liver metastasis and survival outcome in patients with cancer (10–13). One study found that HBV infection significantly decreased the risk of liver metastasis and prolonged the survival in patients with colorectal cancer (10). Recent study showed that HBV infection was significantly associated with more liver metastasis and larger-sized liver metastasis in patients with colorectal liver–only metastasis (CRLM). However, HBV-infected CRLM patients survived longer than noninfected patients after liver resection (13). Moreover, several studies showed that HBV infection was an independent prognostic factor for survival in many cancers (12, 14, 15). Recent study found that hepatitis B virus X-interacting protein, originally identified for interacting with hepatitis B virus X proteins, was high expressed in esophageal squamous cell carcinoma (ESCC) and was an independent and unfavorable prognostic marker for patients with ESCC (16).

Nevertheless, no study to date has investigated the role of HBV infection in the clinical features and survival of patients with esophageal cancer. Given that both esophageal cancer and HBV are endemic in China, we embarked on this study to get insight into the prevalence of HBV infection in patients with esophageal cancer, and evaluate the association of HBV infection with the characteristics and prognosis in esophageal cancer.

We identified consecutive patients with esophageal cancer who underwent surgical resection at Sun Yat-sen University Cancer Center (Guangzhou, China) between December 2000 and December 2008 (17, 18). All patients were newly confirmed to have esophageal cancer and had not received treatment. Patients were excluded on the basis of the following criteria: history of other cancer; prior neoadjuvant therapy; died in the perioperative period; lack of information on HBV infection or hepatitis viral infections other than HBV. Esophagectomy and pathologic stage was described in our previous study (17, 18). All patients provided written informed consent for their information to be stored and used in the hospital database. Study approval was obtained from independent ethics committees at Sun Yat-Sen University Cancer Center (Guangzhou, China).

Blood samples were collected at the first visit of all the patients, and serum samples were separated for the test of HBV infection. ELISA was applied to the tests of HBsAg hepatitis B surface antibody, hepatitis B e antigen, hepatitis B e antibody, and hepatitis B core antibody. In addition, all patients were also tested for serum human immunodeficiency virus antibody, hepatitis A virus antibody, hepatitis C virus antibody, hepatitis D virus antigen, hepatitis D virus antibody, and hepatitis E virus antibody. All assays were performed blinded to the study endpoint. However, the HBV viral loads were not routinely determined. Chronic HBV infection was defined as HBsAg seropositivity.

Standardized follow-up was described in our previous study (18). Follow-up continued until June 2013. The median follow-up time was 32.1 months. Overall survival (OS) and disease-free survival (DFS) are the endpoints as described previously in our study (18).

Statistical analysis was performed using SPSS 16.0 for Windows software system (SPSS Inc). χ² test was performed to evaluate the associations between clinicopathologic variables and HBV infection, respectively. Survival curves and multivariate analysis were performed as described previously in our study (17). We tested the proportional hazards assumption by Schoenfeld residual test and found the test is not statistically significant for each of the covariates as well as the global test. Therefore, we can assume the proportional hazards. A significant difference was declared if the P value from a two-tailed test was less than 0.05.

A total of 2,004 consecutive patients with esophageal cancer were included in the study. Of the patients, 253 (12.6%) were seropositive for HBsAg (HBV infection) and 1,751 (87.4%) were HBsAg-negative. The baseline characteristics of these patients are summarized in Table 1. No significant difference was noted in age, gender, histopathology, smoking status, radicality of surgery, differentiation, tumor location, and lymph node metastasis between HBsAg-positive patients and HBsAg-negative patients. However, HBsAg-positive group had a higher percentage of never alcohol consumption (74.3% vs. 68.5%, P = 0.033) and early pathologic T stage (39.5% vs. 26.3%, P = 0.002) than HBsAg-negative. Distant metastasis after surgery occurred in 183 patients (9.1%), including liver metastasis patients (57; 2.8%) and extrahepatic metastasis patients (126; 6.3%). Interestingly, we found that HBsAg-positive patients had a lower frequency of liver metastasis (0.8% vs. 3.1%, P = 0.036) and extrahepatic metastasis (3.2% vs 6.7%, P = 0.028) when compared with HBsAg-negative patients.

Up to the last day of follow-up, 139 of 253 patients in the HBsAg-positive group (54.9%) and 1,138 of 1,751 patients in the HBsAg-negative group (65.0%) died. Univariate survival analysis showed that patients with HBsAg-positive status had a significantly better DFS (mean:78.7 months vs. 53.3 months, P = 0.002; Table 2; Fig. 1A) and OS (mean:83.6 months vs. 58.6 months, P = 0.009; Table 2; Fig. 1B) than those with HBsAg-negative. As shows in Table 2, male patients, a history of smoking, alcohol consumption, poor histologic differentiation, and advanced pathologic stage III–IV were found to have significantly shorter DFS and OS (P < 0.05). However, no significant association between histopathology, age, or tumor location with DFS or OS was observed in patients with esophageal cancer.

The Cox proportional hazards regression suggested that HBV infection was an independent favorable prognostic factor in operable esophageal cancer (Table 3). In the final multivariate survival analysis with adjustment for prognostic factor, we found that HBsAg-positive patients had 20% and 21% lower risks of disease progression [HR = 0.79, 95% confidence interval (CI): 0.66–0.94, P = 0.007] and death (HR = 0.80; 95% CI:0.65–0.95, P = 0.020) respectively, than HBsAg-negative patients.

Univariate survival analyses stratified by histology, age, gender, smoking status, alcohol consumption, and pathologic stage were performed. We found that HBsAg-positive status was associated with favorable DFS and OS in patients with ESCC (Fig. 2A and B), young age (Fig. 2C and D), male gender (Fig. 2E and F), never smoking (Fig. 3A and B), never alcohol consumption (Fig. 3C and D), and advanced pathologic stage (III–IV; Fig. 3E and F; P < 0.05; Table 4). HBsAg-positive female patients had a better OS than HBsAg-negative patients (P = 0.025), but not for DFS. In addition, the association between HBsAg-positive status and better DFS was observed in patients with ever smoking (P = 0.023). However, there was no significant association between HBV infection and DFS or OS in patients with adenocarcinoma, old age, alcohol consumption, or early pathologic stage (I–II; P > 0.05).

HBV infection has been proven to be associated with many cancers and serves as a prognostic factor to predict the survival outcome of patients (12, 14, 15). Recent study showed that the oncoprotein hepatitis B virus X-interacting protein was highly expressed in ESCC and was an unfavorable independent prognostic marker for patients with ESCC (16). However, the prognostic value of HBV infection in long-term survival of patients with esophageal cancer after esophagectomy has not yet been evaluated. In our study, HBV infection was closely associated with early pathologic T stage. In addition, the incidence of liver metastasis and extrahepatic metastasis after surgery was less frequent in patients with HBsAg-positive in comparison with HBsAg-negative patients. More importantly, HBV infection was found to be an independent favorable prognostic factor in operable esophageal cancer. Patients with HBsAg-positive status had a better DFS and OS than those with HBsAg-negative. To our knowledge, this study is the first large-scale study to determine the impact of HBV infection on the prognosis of patients with esophageal cancer from a population with a high prevalence of both HBV infection and esophageal cancer.

In this study, the HBsAg seropositivity rate in patients with esophageal cancer was 12.6%, which was in accordance with that of the general population in this area (8%–15%; ref. 5). We found that HBV infection was associated with early pathologic T stage, which suggested that HBV infection may play an important role in the progression of esophageal cancer. Previous study reported that the invasive capability of hepatocellular carcinomas can be reduced by HBV infection (19). Besides, HBV replication can promote the production of TNFα by residual immune cells as well as hepatocytes in the liver, which can exhibit an antitumor effect on tumor cells (20). However, the in vivo and in vitro assay as well as the underlying mechanism need to be further studied. Moreover, we demonstrated that HBsAg-positive patients are less likely to develop liver metastasis and extrahepatic metastasis after esophagectomy than HBsAg-negative patients. To date, this study was the first report to demonstrate the influence of HBV infection on metastatic pattern in esophageal cancer. Previous studies investigated the impact of HBV infection on metastatic patterns of other malignancies. Song and colleagues reported that the incidence of liver metastasis in patients with colorectal cancer with HBV infection was significantly lower than that of the noninfection (10). What's more, HBV infection was investigated to decrease the incidence of liver metastasis and increase the incidence of extrahepatic metastasis in patients with colorectal cancer (11). Recent study evaluated the role of HBV infection in patients with colorectal liver–only metastases after liver resection, and found that HBV infection was associated with more liver metastasis and larger-sized liver metastasis. In addition, HBV-positive patients are less likely to develop postoperative recurrence after liver resection than HBV-negative patients and that the former are less likely to develop intrahepatic recurrence than the latter (13). However, one previous study found that incidence of synchronous liver metastasis was significantly higher in patients with pancreatic cancer with HBsAg-positive than those with HBsAg-negative (12). Thus, it can be concluded that the impact of HBV infection on liver metastasis vary in different malignancies. The reasons why HBV-positive patients rarely metastasize to the liver remains unclear, but some studies have been investigated. Budhu and colleagues reported that the hepatic microenvironment in patients with HBV-positive metastatic liver cancer can greatly change their gene expression profiles, and the two significant clusters in the profile indicated notable changes associated with gene products involved in immune function 21). Another study on tumor and stroma interaction suggested that the propensity of metastatic liver cancer is inherent to the tumor cells and affected by the local environment of metastatic sites (22). Therefore, it was reasonable to postulate that HBV infection activates the liver-associated immunity to decrease the incidence of liver metastasis in patients with cancer.

We found that HBsAg-positive patients had a favorable DFS and OS when compared with HBsAg-negative patients. Moreover, multivariate analysis revealed that HBV infection was an independent favorable prognostic factor for DFS and OS in operable esophageal cancer. HBsAg-positive patients had 20% and 21% lower risks of death and disease progression than HBsAg-negative patients. Our findings are similar to previous studies showing that HBV infection prolonged the survival of patients (10, 13). However, other studies revealed that HBV infection was associated with poor survival and served as an independent adverse prognostic factor in patients (14, 15). Our subgroup analysis found that HBsAg-positive status was associated with favorable DFS and OS in patients with ESCC and advanced pathologic stage (III–IV). However, for patients with adenocarcinoma and early pathologic stage (I–II; P > 0.05), there was no significant association between HBV infection and DFS or OS. These findings suggested that HBV infection might serve as a useful biomarker to predict survival in patients with esophageal cancer, especially patients with advanced pathologic stage ESCC. A prospective study is required to determine the prognostic and treatment value of HBV infection in esophageal cancer.

We acknowledged that there are a number of limitations to our study. First of all, although large number of populations was included, our study was a retrospective study from a single-institution experience, which may lead to selection bias. Second, information on posttreatment recurrence was insufficient, which might affect the survival of patients. Third, the data of hepatic function was insufficient and we did not compare the incidence of hepatic adverse event in HBsAg-positive and HBsAg-negative patients. Fourth, we did not analyze the effect of HBV DNA copy number on survival of patients with esophageal cancer because of a lack of data regarding HBV DNA load and HBV replication. Last but not the least, although we conducted subgroup analysis stratified by some prognostic factors with important clinical significance, some findings with P value were near 0.05 without adjustments to the level of statistical significance due to multiple hypothesis testing. Therefore, further prospective studies of large cohorts of patients are warranted to confirm these results.

In conclusion, our study provided definite and quantitative evidence that HBV infection was an independent favorable prognostic factor in patients with esophageal cancer. Further prospective studies of large cohorts of patients that include an analysis of HBV DNA load are warranted to confirm these results. In addition, the latent mechanism by which chronic HBV infection leads to better survival outcomes in patients with esophageal cancer needs to be investigated.

No potential conflicts of interest were disclosed.

Conception and design: J.Y. Zou, Q. Liu, J. Wen, S. Zhang

Development of methodology: J.Y. Zou, X. Cai

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Chen, X. Xie, Z.-G. Liu, J. Wen

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Chen, X. Xie, Z.-G. Liu, X. Cai, Q. Liu

Writing, review, and/or revision of the manuscript: J. Chen, X. Xie, Q. Liu, S. Zhang

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): X. Xie, J. Wen

Study supervision: S. Zhang

This work was supported by grants from the Science and Technology Planning Project of Guangdong Province, China (A2016042) and Wu Jieping Medical Foundation (320.320.2730.1875; to S. Zhang), National Science Foundation of China (81672356), Guangzhou Science Technology and Innovation Commission (201610010127), and Guangdong Talents Special Support Program (201629038; to J. Wen). The authors would like to thank all patients without whom this study would not have been possible.

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