Chronic Hepatitis B Virus Infection And Extra-liver Cancer Risk:Prospective Studies From Chinese Populations

[Background]Hepatitis B virus(HBV)infection is one of the most serious and prevalent health problems,affecting more than 2 billion people worldwide.There are 700 to 800 million people infected with HBV in China.Chronic HBV infection includes asymptomatic and chronic hepatitis B carriers,both of which are closely related to cirrhosis and hepatocellular carcinoma(HCC).HBV infection would lead to acute and chronic liver diseases,including acute or chronic hepatitis B,cirrhosis or hepatocellular carcinoma(HCC).HBV has been implicated in the etiology of up to 80%of liver cancer,which frequently occurs in Chinese and African populations.The virus optimizes its lifecycle to allow for long-term persistence in liver tissue by establishing a plasmid-like covalently closed circular DNA(cccDNA)form.Chronic HBV infection persisting in liver tissue is associated with increased chronic oxidative damage in hepatocytes,immune-mediated inflammation of the liver and cancer development in patients.A few clinical case studies detected HBV in several types of extrahepatic tissues,suggesting a potential role of HBV in the oncogenesis of extrahepatic cancers.Few population-based prospective studies have found associations between chronic HBV infection and various extrahepatic cancers,but these findings have been inconsistent.A few prospective studies have examined the association between HBV infection and extrahepatic cancers.Overall,the majority of previous studies revealed that HBV was associated with pancreatic cancer and lymphoma.However,a European cohort proposed that pancreatic cancer and lymphoma were not higher in the HBV-infected cohort compared to non-HBV infected individuals.Most of these studies were based on existing health or hospital registries or health insurance claim data.The lack of detailed individual information led to minimal control of potential confounding.Hospital-based identification of participants might also overestimate the extrahepatic cancer incidence of HBV carriers.Furthermore,previous population-based studies used the level of HBV infection markers in peripheral blood,which represents the degree of virus replication in the liver instead of that in the extrahepatic tissues.Therefore,whether the observed association between HBV infection and extrahepatic cancers was due to the general influence of the virus on the immune system or specific attacks on the extrahepatic histocytes remains unclear.In order to overcome the shortcomings of the above research design,many authoritative epidemiologists have proposed that population based prospective cohort will play a vital important role in the etiology study area of human chronic disease.The population based prospective cohort studies have many advantages including better study efficiency,higher repeatability,more credibility,etc.In addition,they could make up the limitation of small scale cohort studies that it is difficult to obtain cases or enough cases of rare diseases.Hence,we aimed to examine the associations between chronic HBV infection and extrahepatic cancer.[Method]In the present study,we aimed to examine the associations between chronic HBV infection and extrahepatic cancer using three substudies with complementary advantages.We first analyzed the associations between HBV infection and liver and extrahepatic cancer in the China Kadoorie Biobank study of 0.5 million adults,in which HBV infection was detected using an on-site HBsAg rapid test(substudy 1).To minimize the potential influence of infection misclassification on effect estimation,we replicated the association analysis in the Qidong prospective cohort and a nested case-control study established within the Changzhou cohort,in both of which more sensitive detection assays for HBsAg were employed(substudy 2).Finally,we examined whether there existed HBV replication and expression in the extrahepatic cancer tissues of the patients with stomach cancer,pancreatic cancer,and lung cancer(substudy 3).The discovery phase is based on the China Kadoorie Biobank(CKB,substudy 1),a prospective cohort study of over 0.5 million adults.After four years of baseline survey,a total of 515,681 participants participated in the collection of baseline information of the CKB project.After quality control,a total of 512,891 participants were included in the follow-up analysis.We them excluded 11,733 participants who had missing data or unclear results for HBsAg,2,522 participants with cancer at baseline,and 1,904 participants who developed cancer during the first year of follow-up.A total of 496,732 participants were included in the final analysis.The start date of follow-up was the date of the baseline survey completed.The outcome of follow-up was cancer onset.The termination time of follow-up was December 31,2015.Considering the low sensitivity of the on-site HBsAg rapid test used in the CKB cohort,we replicated the analysis in two other studies using more sensitive detection assays.A population-based cohort study was established from 2007 to 2011 in Qidong County,Jiangsu Province(substudy 2).In the six randomly selected villages,inhabitants 30 to 70 years of age who had been living in their current residence for at least five years were eligible to participate.A total of 37,927 participants provided written informed consent for the interview and blood collection for serological assays.In the present analysis,we excluded participants who reported a prior history of cancer at baseline(n=372)and those who developed cancer during the first year of follow-up(n=219),leaving 37,336 participants for analysis.The start date of follow-up was the date of the baseline survey completed.The outcome of follow-up was cancer onset.The termination time of follow-up was December 31,2015,A population-based cohort was established between 2004 and 2005 in the Wujin District in Changzhou C ity,Jiangsu Province.Overall,17,723 participants aged 35 years or older who had been living in their current residence for at least five years were eligible to participate.After follow-up,the cohort resulted in the identification of 141 new cases of stomach cancer;118 of these patients provided sufficient blood samples.We then performed a nested case-control study in which 118 stomach cancer cases were matched to 472 healthy controls(1:4)on the basis of age and sex(Figure 1).In the CKB and Qidong cohort,we used Cox regression to estimate the hazard ratios(HRs)and their 95%confidence intervals(95%CIs)with adjustment for baseline covariates.In the CKB cohort,the multivariate adjusted HRs(95%CIs)of cancer were estimated with the following adjustments:age,sex,residential area,level of education,marital status,family income,alcohol consumption,smoking status,BMI,and family history of cancer.The Kaplan-Meier method and log-rank test were used to compare the cancer onset age in different subgroups categorized by HBsAg status.In the Qidong cohort,the multivariate adjusted HRs(95%CIs)of cancer were estimated with age and gender.For the analysis of the Changzhou nested case-control study,we performed logistic regression analyses to calculate odds ratios(ORs)and their 95%CIs.The multivariate adjusted ORs(95%CIs)of cancer were estimated with the following adjustments:age,sex,residential area,level of education,marital status,family income,alcohol consumption,smoking status,BMI,and family history of cancer.The on-site HBsAg rapid test was simple-used,rapid,and suitable for large sample population.However,considering the low sensitivity of the on-site HBsAg rapid test used in the CKB cohort,we replicated the analysis in two other studies using more sensitive detection assays.To explain the potential associations between HBV infection and extrahepatic cancer,we then detected HBV replication and expression in extrahepatic cancer tissues.The surgically resected tissues of primary stomach cancer were collected from(1)Nantong Tumor Hospital(Nantong,Jiangsu,China)and First Affiliated Hospital of Suzhou University(Suzhou,Jiangsu,China)between 2014 and 2015(n=47);(2)Zhejiang Tumor Hospital(Hangzhou,Zhejiang,China)between 2011 and 2012.HBV replication and cccDNA were identified by PCR.Stomach cancer cases from both Jiangsu and Zhejiang showed similar distributions of serum anti-HBc and HBV DNA in the stomach cancer tissues.Next,cccDNA amplicons from the 12 HBV DNA positive Jiangsu cancer cases were sequenced.Moreover,of the 12 HBV DNA positive Jiangsu stomach cancer cases,eight formalin-fixed paraffin-embedded cancer tissue sections were collected to examine the HBX and anti-HBc protein expression using IHC staining.[Results]In substudy 1,the prevalence of HBsAg positivity was 3.1%.HBsAg-positive participants were more likely to be younger,male,urban and coastal residents.During 4.4 million person-years of follow-up,there were 20,891 incident cancer cases.Multivariable-adjusted analyses showed that HBsAg seropositivity was associated with an increased risk of overall cancer incidence(HR,95%CI:2.18,2.05-2.32).HBsAg seropositivity was also associated with the risks of several site-specific cancers,including liver cancer(HR,95%CI:15,77,14.15-17.57),stomach cancer(1.41,1.11-1.80),colorectal cancer(1.42,1.12-1.81),oral cancer(1.58,1.01-2.49),lymphoma(2.10,1.34-3.31),and pancreas cancer(1.65,1.03-2.65).There were no statistically significant associations between HBsAg seropositivity and other cancer sites.We further examined the potential influence of HBV infection on the age at cancer onset among cancer cases.HBsAg seropositive cases had an earlier age of onset of liver cancer,oral cancer,colorectal cancer,and pancreatic cancer,compared with HBsAg seronegative cases(P<0.001,0.003,0.006,0.034).This trend was not observed for stomach cancer and lymphoma(P=0.008,0.016).Of 37,336 participants from the Qidong cohort,9.5%were HBsAg seropositive.During 255,752 person-years of follow-up,there were 1,386 incident cancer cases documented(data not shown).Age-and sex-adjusted Cox regression analysis showed that HBsAg seropositivity was also associated with an increased risk of overall cancer,with an HR(95%CI)of 3.39(2.95-3.89).Similarly,the risks of developing liver cancer(HR,95%CI:17.51,13.86-22.11)and stomach cancer(2.02,1.24-3.29)were higher in HBsAg seropositive than in HBsAg seronegative participants.In the study population,there were only five colorectal cancer cases among HBsAg-positive participants,and no cases of oral cancer,pancreatic cancer or lymphoma reported.In the results,we also found an association of HBsAg seropositivity with the risk of stomach cancer(OR=1.76,95%CI=1.04-2.98,P=0.035).Among the Jiangsu and Zhejiang cases,46.8%(22/47)and 70.0%(35/50)were anti-HBc seropositive,respectively.Furthermore,among those anti-HBc seropositive stomach cancer cases,the prevalence of HBV DNA positivity in cancer tissues was 54.5%(12/22)and 54.3%(19/35),respectively.In contrast,none of the HBV DNA was detected in the anti-HBc seronegative cancer cases.After the PCR products were sequenced and aligned,cccDNA was found in one Jiangsu stomach cancer tissue sample with positive HBV DNA,suggesting that HBV could actively replicate in stomach tissue but not induce HBsAg positivity in the serum.All of the collected cancer cases were anti-HBc seropositive.HBX and anti-HBc proteins were highly expressed in the stomach cancer cells of all the stomach cancer tissue sections,but they were minimally expressed in the normal parts of the specimens.The same phenomenon was observed in all of the ten pancreatic cancer sections,the representative HBV-related cancer identified in the CKB cohort.However,we did not find the HBX or anti-HBc proteins expressed in the nine lung cancer cells or normal lung epithelial cells of any of the lung cancer sections,which was also consistent with the findings from the CKB cohort.[Conclusion]In a large prospective Chinese cohort of 0.5 million adults,we found that HBsAg seropositive participants were at an increased risk of developing both liver cancer and several extrahepatic cancers,including stomach cancer,oral cancer,colorectal cancer,pancreatic cancer,and lymphoma.The association between HBsAg seropositivity and stomach cancer was further replicated in two other small Chinese studies,in which more sensitive assays for HBsAg detection were used.Our tissue-based experiments validated the occurrence of HBV expression in cancer cells located in the stomach and pancreas,but not in lung cancer cells.Our study provides compelling evidence that HBV infection increases extrahepatic cancer risks,especially for digestive system cancers.It highlights the importance of early screening for digestive system cancer in the HBV-infected population.