Qidong: a crucible for studies on liver cancer etiology and prevention

Qidong(Jiangsu, China) has been of interest to cancer epidemiologists and biologists because, until recently, it was an endemic area for liver cancer, having amongst the highest incidence rates in the world. The establishment of the Qidong Cancer Registry together with the Qidong Liver Cancer Institute in 1972 has charted the patterns of liver cancer incidence and mortality in a stable population throughout a period of enormous economic, social, and environmental changes as well as of improvements in health care delivery. Updated incidence trends in Qidong are described. Notably, the China age-standardized incidence rate for liver cancer has dropped by over 50% in the past several decades. Molecular epidemiologic and genomic deep sequencing studies have affirmed that infection with hepatitis B virus as well as dietary exposure to aflatoxins through contamination of dietary staples such as corn, and to microcystins–blue-green algal toxins found in ditch and pond water – were likely important etiologic factors that account for the high incidence of liver cancer in this region. Public health initiatives to facilitate universal vaccination of newborns against HBV and to improve drinking water sources in this rural area, as well as economic and social mandates serendipitously facilitating dietary diversity, have led to precipitous declines in exposures to these etiologic factors, concomitantly driving substantive declines in the liver cancer incidence seen now in Qidong. In this regard, Qidong serves as a template for the global impact that a package of intervention strategies may exert on cancer burden.

Long-term survival trends of gastric cancer patients between 1972 and 2011 in Qidong

Background:There have been few reports on long-term survival of gastric cancer patients.This study analyzed the survival data of gastric cancer patients obtained from the population-based Qidong Cancer Registry between 1972 and 2011,providing a basis for evaluation of gastric cancer treatment and prognosis.Methods:The cumulative observed survival rate and relative survival rate of gastric cancer patients were calculated using Hakulinen's method via the SURV3.01 software,which was developed by the Finnish Cancer Registry.The date of the last follow-up for the survival status of the 15,401 registered cases was April 30,2012.Results:The 1-,5-,10-,20-,and 30-year observed survival rates were 33.82%,14.18%,10.35%,6.63%,and 4.19%,respectively,and the 1-,5-,10-,20-,and 30-year relative survival rates were 35.43%,18.13%,17.50%,21.96%,and32.84%,respectively.For males,the corresponding observed survival rates were 34.50%,14.40%,10.42%,6.46%,and4.05%,and the corresponding relative survival rates were 36.23%,18.67%,18.28%,23.73%,and 38.61%.For females,the corresponding observed survival rates were 32.62%,13.80%,10.22%,6.95%,and 4.46%,and the corresponding relative survival rates were 34.03%,17.21%,16.28%,19.70%,and 26.78%.Significant differences in relative survival rates were observed between sexes(P=0.003).For the 15-34,35-44,45-54,55-64,65-74,and 75+ age groups,the 5-year relative survival rates were 16.13%,21.77%,18.63%,12.61%,7.99%,and 2.94%,respectively,and the 10-year relative survival rates were 16.49%,22.83%,20.50%,15.97%,15.88%,and 15.73%,respectively.Remarkable improvement could be observed for the 5-,10-,and 15-year relative survival rates in Qidong beginning in the 1980 s.Conclusion:The survival outcome of registered gastric cancer cases in Qidong showed gradual progress over the past two decades.

Suppression of Human Liver Cancer Cell Migration and Invasion via the GABA_A Receptor

Objective To investigate the roles of theγ-aminobutyric acid(GABA) in the metastasis of hepatocellular carcinoma(HCC) and to explore the potential of a novel therapeutic approach for the treatment of HCC. Methods The expression levels of GABA receptor subunit genes in various HCC cell lines and patients’ tissues were detected by quantitative real-time polymerase chain reaction and Western blot analysis.Transwell cell migration and invasion assays were carried out for functional analysis.The effects of GABA on liver cancer cell cytoskeletal were determined by immunofluorescence staining. And the effects of GABA on HCC metastasis in nude mice were evaluated using an in vivo orthotopic model of liver cancer. Results The mRNA level of GABA receptor subunits varied between the primary hepatocellular carcinoma tissue and the adjacent non-tumor liver tissue.GABA inhibited human liver cancer cell migration and invasion via the ionotropic GABAa receptor as a result of the induction of liver cancer cell cytoskeletal reorganization.Pretreatment with GABA also significantly reduced intrahepatic liver metastasis and primary tumor formation in vivo. Conclusions These findings introduce a potential and novel therapeutic approach for the treatment of cancer patients based on the modulation of the GABAergic system.

A case-control study of the relationship between hepatitis B virus DNA level and risk of hepatocellular carcinoma in Qidong,China

AIM:To investigate the role of hepatitis B virus (HBV) replication in the development of hepatocellular carcinoma (HCC), a nested case-control study was performed to study the relationship between HBV DNA level and risk of HCC. METHODS:One hundred and seventy cases of HCC and 276 control subjects free of HCC and cirrhosis were selected for this study. Serum HBV DNA level was measured using fluorescein quantitative polymerase chain reaction at study entry and the last visit. RESULTS:In a binary unconditional logistic regression analysis adjusted for age, cigarette smoking, alcohol consumption and family history of chronic liver diseases, the adjusted odds ratios (95% confidence intervals) of HCC in patients with increasing HBV DNA level were 2.834 (1.237-6.492), 48.403 (14.392-162.789), 42.252 (14.784-120.750), and 14.819 (6.992-31.411) for HBV DNA levels ≥ 104 to < 105; ≥ 105 to < 106; ≥ 106 to < 107; ≥ 107 copies/mL, respectively. Forty-six HCC cases were selected to compare the serums viral loads of HBV DNA at study entry with those at the last visit. The HBV DNA levels measured at the two time points did not differ significantly.CONCLUSION:The findings of this study provide strong longitudinal evidence of an increased risk of HCC associated with persistent elevation of serum HBV DNA level in the 104-107 range.

Qidong hepatitis B virus infection cohort: a 25-year prospective study in high risk area of primary liver cancer

Qidong hepatitis B virus (HBV) infection cohort (QBC) is a prospective community-based study designed to investigate causative factors of primary liver cancer (PLC) in Qidong, China, where both PLC and HBV infection are highly endemic. Residents aged 20-65 years, living in seven townships of Qidong, were surveyed using hepatitis B surface antigen (HBsAg) serum test and invited to participate in QBC from June 1991 to December 1991. A total of 852 and 786 participants were enrolled in HBsAg-positive and HBsAg-negative sub-cohorts in May 1992, respectively. All participants were actively followed up in person, received HBsAg, alanine aminotransferase, alpha-fetoprotein tests and upper abdominal ultrasonic examination, and donated blood and urine samples once or twice a year. The total response rate was 99.6%, and the number of incident PLC was 201 till the end of February 2017. The ratio of incidence rates was 12.32 [95% confidence interval (CI): 7.16-21.21, P < 0.0001] in HBsAg-positive arm compared with HBsAg-negative arm. The relative risk of PLC was 13.25 (95%CI: 6.67-26.33, P < 0.0001) and 28.05 (95% CI: 13.87-56.73, P < 0.0001) in the HBsAg+/HBeAg-group and the HBsAg+/HBeAg+ group, respectively, as compared to the HBsAg-/HBeAg- group. A series of novel PLC-related mutations including A2159G, A2189C and G2203W at the C gene, A799G, A987G and T1055A at the P gene of HBV genome were identified by using samples from the cohort. The mutation in HBV basal core promoter region of HBV genome has an accumulative effect on the occurrence of PLC. In addition, the tripartite relationship of aflatoxin exposure, P53 mutation and PLC was also investigated. QBC will be used to develop dynamic prediction model for PLC risk by using its long-term follow-up information and serial blood samples. This model is expected to improve the efciency of PLC screening in HBV infection individuals.

Role of transforming growth factor-betal-smad signal transduction pathway in patients with hepatocellular carcinoma

AIM:To explore the role of transforming growth factor-beta1(TGF-β1)-smad signal transduction pathway inpatients with hepatocellular carcinoma.METHODS:Thirty-six hepatocellular carcinoma speci-mens were obtained from Qidong Liver Cancer Instituteand Department of Pathology of the Second AffiliatedHospital of Nanjing Medical University.All primary anti-bodies(polyclonal antibodies)to TGF-β1,type Ⅱ Trans-forming growth factor-beta receptor(TβR-Ⅱ),nuclear fac-tor-kappaB(NF-KB),CD34,smad4 and smad7,secondaryantibodies and immunohistochemical kit were purchasedfrom Zhongshan Biotechnology Limited Company(Bei-jing,China).The expressions of TGF-β1,TβR-Ⅱ,NF-kB,smad4 and smad7 proteins in 36 specimens ofhepatocellular carcinoma(HCC)and its adjacent tissuewere separately detected by immunohistochemistry toobserve the relationship between TGF-β1 and TβR-Ⅱ,between NF-kB and TGF-β1,between smad4 and smad7and between TGF-β1 or TβR-Ⅱ and microvessel density(MVD).MVD was determined by labelling the vesselendothelial cells with CD34.RESULTS:The expression of TGF-β1,smad7 and MVDwas higher in HCC tissue than in adjacent HCC tissue(P<0.01,P<0.05,P<0.01 respectively).The expressionof TβR-Ⅱ and smad4 was lower in HCC tissue than in its adjacent tissue(P<0.01,P<0.05 respectively).Theexpression of TGF-β1 protein and NF-kB protein wasconsistent in HCC tissue.The expression of TGF-β1 andMVD was also consistent in HCC tissue.The expressionof TβR-Ⅱ was negatively correlated with that of MVD inHCC tissue.CONCLUSION:The expressions of TGF-β1,TβR-Ⅱ,NF-kB,smad4 and smad7 in HCC tissue,which are ma-jor up and down stream factors of TGF-β1-smad signaltransduction pathway,are abnormal.These factors areclosely related with MVD and may play an important rolein HCC angiogenesis.The inhibitory action of TGF-β1 isweakened in hepatic carcinoma cells because of abnor-mality of TGF-β1 receptors(such as TβR-Ⅱ)and postre-ceptors(such as smad4 and smad7).NF-kB may causeactivation and production of TGF-β1.

Novel approach to identifying the hepatitis B virus pre-S deletions associated with hepatocellular carcinoma

AIM: To develop a novel non-sequencing method for the detection of hepatitis B virus(HBV) pre-S deletion mutants in HBV carriers.METHODS: The entire region of HBV pre-S1 and pre-S2 was amplified by polymerase chain reaction(PCR). The size of PCR products was subsequently determined by capillary gel electrophoresis(CGE). CGE were carried out in a PACE-MDQ instrument equipped with a UV detector set at 254 nm. The samples were separated in 50 μm ID eCAP Neutral Coated Capillaries using a voltage of 6 kV for 30 min. Data acquisition and analysis were performed using the 32 Karat Software. A total of114 DNA clones containing different sizes of the HBV pre-S gene were used to determine the accuracy of the CGE method. One hundred and fifty seven hepatocellular carcinoma(HCC) and 160 non-HCC patients were recruited into the study to assess the association between HBV pre-S deletion and HCC by using the newly-established CGE method. Nine HCC cases with HBV pre-S deletion at the diagnosis year were selected to conduct a longitudinal observation using serial serum samples collected 2-9 years prior to HCC diagnosis.RESULTS: CGE allowed the separation of PCR products differing in size > 3 bp and was able to identify 10% of the deleted DNA in a background of wild-type DNA. The accuracy rate of CGE-based analysis was 99.1% compared with the clone sequencing results. Using this assay, pre-S deletion was more frequently found in HCC patients than in non-HCC controls(47.1% vs 28.1%, P < 0.001). Interestingly, the increased risk of HCC was mainly contributed by the short deletion of pre-S. While the deletion ≤ 99 bp was associated with a 2.971-fold increased risk of HCC(95%CI: 1.723-5.122, P < 0.001), large deletion(> 99 bp) did not show any association with HCC(P = 0.918, OR = 0.966, 95%CI: 0.501-1.863). Of the 9 patients who carried pre-S deletions at the stage of HCC, 88.9%(8/9) had deletions 2-5 years prior to HCC, while only 44.4%4(4/9) contained such deletions 6-9 years prior to HCC.CONCLUSION: CGE is a sensitive approach for HBV pre-S deletion analysis. Pre-S deletion, especially for short DNA fragment deletion, is a useful predictive marker for HCC.

Liver cancer screening in China: practices and its extended questions

Screening for liver cancer(hepatocellular carcinoma)in China started in early 1970s with the application of alpha-fetoprotein(AFP)in high-incidence regions.It has been extended to nationwide areas,emerging from the concepts of conducting screening in populations at-risk with positive hepatitis B surface antigen to the practice programs in rural and urban areas,and finally to the development of recommendations to guide medical practice for health care providers.The implementation of screening for liver cancer has resulted in earlier detection and hence the early curable treatment for patients who have gained short-or long-term survival,and even reduction in mortality rates,although these outcomes are more anecdotal than rigorously evidence-based.AFP or ultrasound examination has been considered as sensitive and specific methods for early detection but are with limitations.The combined use of these two modalities for screening populations at-risk every six months seems to have been reached consensus.The feasibility of screening for liver cancer is still debated because of differing opinions and even opposition to the choice of targeted sub-populations,the intrinsic necessity,and the contributions of the main risk factors among Western countries and China/Asian areas.Yet,the over 51%of global burden of liver cancer is in China,the solution to the early detection and treatment of liver cancer should fully consider the actual situation in China.The effectiveness of screening for liver cancer is worthy of anticipation.

Id4 promotes cell proliferation in hepatocellular carcinoma

Background: Hepatocellular carcinoma(HCC) is a common malignant tumor in the world, especially in China. As a member of the inhibitor of differentiation(Id) family, Id4 has been reported to function in many cancer types, but relatively little is known about its role in HCC. The purpose of this study was to investigate the potential relationship between Id4 and HCC development and the underlying mechanism involving the function of Id4 in HCC.Methods: We used quantitative real?time polymerase chain reaction and Western blotting to examine the RNA and protein expression of Id4. In addition, we used Cell Counting Kit?8 assay and colony formation assay to identify the function of Id4 in the regulation of cell proliferation in human HCC.Results: We found that the expression of Id4 protein was up?regulated in tumor tissues from HCC patients. Over?expression of Id4 promoted HCC cell proliferation, clonogenicity in vitro, and tumorigenicity in vivo. Id4 knockdown experiments showed that silencing Id4 blocked the proliferation and colony formation ability of HCC cells in vitro. Furthermore, overexpression of CCAAT/enhancer?binding protein β inhibited Id4 expression in HCC cells.Conclusion: Id4 may be developed as a potent therapeutic agent for the treatment of HCC, but more details about the underlying mechanisms of action are needed.

Cholangiocarcinoma Arising 38 Years after Surgical Resection of Hepatocellular Carcinoma

Background: Most of the reports on tumor relapse are recurrence of the same type of tumor after months to few years of a successful initial cancer treatment. It is generally unusual and unexpected that a different type of the second tumor occurs after several decades of the curative treatment of the original tumor. Case Presentation: We report a case of 74-year-old man with intrahepatic cholangiocarcinoma (ICC) diagnosed 38 years after curative resection of hepatocellular carcinoma (HCC). In addition to the uniqueness of longer survival and developed a new type of tumor, both the original HCC and the later occurred ICC were detected through a cancer surveillance program by screening alpha-fetoprotein (AFP) and ultrasonography of the liver for the general population and/or high risk group of people who were asymptomatic. Conclusion: This report provides evidence demonstrating occurrence of new type of tumor following initial curative therapy of the original tumor. In addition, this case report also highlights the importance of cancer surveillance program for earlier detection of the tumors to achieve a remarkably improved prognosis of the cancer patients for a prolonged cancer free survival time.

Dynamic edge-based biomarker non-invasively predicts hepatocellular carcinoma with hepatitis B virus infection for individual patients based on blood testing

Hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) is a major cause of cancer-related deaths in Asia and Africa. Developing effective and non-invasive biomarkers of HCC for individual patients remains an urgent task for early diagnosis and convenient monitoring. Analyzing the transcriptomic profiles of peripheral blood mononuclear cells from both healthy donors and patients with chronic HBV infection in different states (i.e. HBV carrier, chronic hepatitis B, cirrhosis, and HCC), we identified a set of 19 candidate genes according to our algorithm of dynamic network biomarkers. These genes can both characterize different stages during HCC progression and identify cirrhosis as the critical transition stage before carcinogenesis. The interaction effects (i.e. coexpressions) of candidate genes were used to build an accurate prediction model: the so-called edge-based biomarker. Considering the convenience and robustness of biomarkers in clinical applications, we performed functional analysis, validated candidate genes in other independent samples of our collected cohort, and finally selected COL5A1, HLA-DQB1, MMP2, and CDK4 to build edge panel as prediction models. We demonstrated that the edge panel had great performance in both diagnosis and prognosis in terms of precision and specificity for HCC, especially for patients with alpha-fetoprotein-negative HCC. Our study not only provides a novel edge-based biomarker for non-invasive and effective diagnosis of HBV-associated HCC to each individual patient but also introduces a new way to integrate the interaction terms of individual molecules for clinical diagnosis and prognosis from the network and dynamics perspectives.