Ground Glass Hepatocytes Research Articles

Characterization of integrated hepatitis B virus DNA harboring pre-S mutations in hepatocellular carcinoma patients with ground glass hepatocytes.

Ground glass hepatocytes (GGHs) have been associated with hepatocellular carcinoma (HCC) recurrence and poor prognosis. We previously demonstrated that pre-S expression in some GGHs is resistant to current hepatitis B virus (HBV) antiviral therapies. This study aimed to investigate whether integrated HBV DNA (iDNA) is the primary HBV DNA species responsible for sustained pre-S expression in GGH after effective antiviral therapy. We characterized 10 sets of micro-dissected, formalin-fixed-paraffin-embedded, and frozen GGH, HCC, and adjacent hepatitis B surface antigen-negative stained tissues for iDNA, pre-S deletions, and the quantity of covalently closed circular DNA. Eight patients had detectable pre-S deletions, and nine had detectable iDNA. Interestingly, eight patients had integrations within the TERT and CCNE1 genes, which are known recurrent integration sites associated with HCC. Furthermore, we observed a recurrent integration in the ABCC13 gene. Additionally, we identified variations in the type and quantity of pre-S deletions within individual sets of tissues by junction-specific PacBio long-read sequencing. The data from long-read sequencing indicate that some pre-S deletions were acquired following the integration events. Our findings demonstrate that iDNA exists in GGH and can be responsible for sustained pre-S expression in GGH after effective antiviral therapy.

Histological Patterns of Hepatitis and Cholangitis

Histological Patterns of Hepatitis and Cholangitis

Ground-glass hepatocellular inclusions are associated with polypharmacy

Ground-glass (GG) hepatocytes are classically associated with chronic hepatitis B (HBV) infection, storage disorders, or cyanamide therapy. In a subset of cases, an exact etiology cannot be identified. In this study, we sought to characterize the clinical, histological, and ultrastructural findings associated with HBV-negative GG hepatocytes. Our institutional laboratory information system was searched from 2000 to 2019 for all cases of ground-glass hepatocytes. Ten liver biopsies with GG hepatocellular inclusions and negative HBV serology, no known history of storage disorders, or cyanamide therapy were reviewed. Half of the patients had history of organ transplantation and/or malignancy. These patients took on average 8.1 medications (range: 3–14) with the most common medications being immunosuppressive and health supplements. Histologically, GG hepatocytes show either peri-portal or centrizonal distribution. The inclusions are PAS-positive and diastase sensitive. Electron microscopy showed intracytoplasmic granular inclusions with low electron density, consistent with unstructured glycogen. In summary, GG hepatocytes are a rare finding in liver biopsies, but are more common in patients with hepatitis B. They can also be seen in HBV-negative patients who have polypharmacy. In these cases, they are the result of unstructured glycogen accumulation putatively due to altered cell metabolism.

Lipid Storage and Interferon Response Determine the Phenotype of Ground Glass Hepatocytes in Mice and Humans.

Background and AimsA histopathological hallmark of chronic hepatitis B virus (HBV) infection is the presence of ground glass hepatocytes (GGHs). GGHs are liver cells that exhibit eosinophilic, granular, glassy cytoplasm in light microscopy and are characterized by accumulation of HBV surface (HBs) proteins in the endoplasmic reticulum (ER). More important, GGHs have been accepted as a precursor of HCC and may represent preneoplastic lesions of the liver.MethodsHere we show that the reason for ground glass phenotype of hepatocytes in patients with chronic hepatitis B (CHB) and in HBs transgenic mice is a complex formation between HBs proteins and lipid droplets (LDs) within the ER.ResultsAs fat is a main component of LDs their presence reduces the protein density of HBs aggregates. Therefore, they adsorb less amount of eosin during hematoxylin-eosin staining and appear dull in light microscopy. However, after induction of interferon response in the liver LDs were not only co-localized with HBs but also distributed throughout the cytoplasm of hepatocytes. The uniform distribution of LDs weakens the contrast between HBs aggregates and the rest of the cytoplasm and complicates the identification of GGHs. Suppression of interferon response restored the ground glass phenotype of hepatocytes.ConclusionsComplex formation between HBs and LDs represents a very important feature of CHB that could affect LDs functions in hepatocytes. The strain specific activation of the interferon response in the liver of HBs/c mice prevented the development of GGHs. Thus, manipulation of LDs could provide a new treatment strategy in the prevention of liver cancer.

Association of the Hepatitis B Virus Large Surface Protein with Viral Infectivity and Endoplasmic Reticulum Stress-mediated Liver Carcinogenesis.

Hepatitis B is the most prevalent viral hepatitis worldwide, affecting approximately one-third of the world’s population. Among HBV factors, the surface protein is the most sensitive biomarker for viral infection, given that it is expressed at high levels in all viral infection phases. The large HBV surface protein (LHBs) contains the integral pre-S1 domain, which binds to the HBV receptor sodium taurocholate co transporting polypeptide on the hepatocyte to facilitate viral entry. The accumulation of viral LHBs and its prevalent pre-S mutants in chronic HBV carriers triggers a sustained endoplasmic reticulum (ER) overload response, leading to ER stress-mediated cell proliferation, metabolic switching and genomic instability, which are associated with pro-oncogenic effects. Ground glass hepatocytes identified in HBV-related hepatocellular carcinoma (HCC) patients harbor pre-S deletion variants that largely accumulate in the ER lumen due to mutation-induced protein misfolding and are associated with increased risks of cancer recurrence and metastasis. Moreover, in contrast to the major HBs, which is decreased in tumors to a greater extent than it is in peritumorous regions, LHBs is continuously expressed during tumorigenesis, indicating that LHBs serves as a promising biomarker for HCC in people with CHB. Continuing efforts to delineate the molecular mechanisms by which LHBs regulates pathological changes in CHB patients are important for establishing a correlation between LHBs biomarkers and HCC development.

Abstract 3630: Comprehensive integrated HBV DNA profiling in HCC patients with GGH by HBV-targeted NGS and PacBio sequencing analysis

Abstract Despite current antiviral therapy success in reducing viral load in patients with chronic hepatitis B virus (HBV), persistent viral surface antigen (HBsAg) remains a risk factor of HCC. Recent evidence has shown that integrated HBV can serve as a source for continued viral antigen expression, despite the lack of detectable replication. Given the oncogenicity of pre-S mutant proteins and its expression in type II ground glass hepatocytes (GGH) even in the absence of viral replication, we assessed the likelihood of GGH as a pre-malignant state as compared to other adjacent-to-tumor non-GGH “normal” tissue by investigating the integrity of integrated HBV DNA. We developed a sensitive HBV-targeted NGS and downstream bioinformatics pipeline, ChimericSeq, to identify HBV-host junctions. A pilot study of 10 HBV-HCC patients, each with six micro-dissected FFPE tissue sections (2 tumor, 2 GGH, 2 HBsAg-negative), was subjected to HBV-targeted NGS and ChimericSeq analysis. We identified integrated HBV DNA in 90% of HCC as well as its matched GGH, and identified PreS2 deletions, for instance, deletion at nt. 43-52. Surprisingly, the recurrent insertional driver mutations of TERT, CCNE1, and ABCC13 integration were detected in 9 of 10 (90%) of this GGH/HCC patient cohort. Next we performed PacBio amplicon sequencing of integrated HBV DNA including Pre-S sequences to comprehensively profile the integrity of the integrated HBV DNA, such as insertional mutations or deletion, and mutations across the different frozen tissue sections (tumor, GGH, HBsAg-negative). Our data suggest that HBV integration of cancer driver genes occurs early during course of infection and integrated DNA evolved over time during carcinogenesis. Overall, a sensitive workflow to comprehensively profile integrated HBV DNA in HCC patients enables further investigation of the role of HBV integration during tumorigenesis. Citation Format: Yu-Lan Kao, Yih-Ping Su, Hung-Wen Tsai, Ih-Jen Su, Selena Lin, Wei Song, Ying-Hsiu Su. Comprehensive integrated HBV DNA profiling in HCC patients with GGH by HBV-targeted NGS and PacBio sequencing analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3630.

Hepatic histopathological and ultrastructural alterations induced by 10 nm silver nanoparticles.

Silver nanoparticles (Ag NPs) are invested in various sectors and are becoming more persistent in our ambient environment with potential risk on our health and the ecosystems. The current study aims to investigate the histological, histochemical and ultrastructural hepatic changes that might be induced by 10 nm silver nanomaterials. Male mice (BALB/C) were exposed for 35 injections of daily dose of 10 nm Ag NPs (2 mg/kg). Liver tissues were subjected to examination by light and electron microscopy for histological, histochemical and ultrastructural alterations. Exposure to Ag NPs induced Kupffer cells hyperplasia, sinusoidal dilatation, apoptosis, ground glass hepatocytes appearance, nuclear changes, inflammatory cells infiltration, hepatocytes degeneration and necrosis. In addition, 10 nm Ag NPs induced histochemical alterations mainly glycogen depletion with no hemosiderin precipitation. Moreover, these nanomaterials exhibited ultrastructure alterations including mitochondrial swelling and cristolysis, cytoplasmic vacuolation, apoptosis, multilammellar myelin figures formation and endoplasmic destruction and reduction. The findings revealed that Ag NPs can induce alterations in the hepatic tissues, the chemical components of the hepatocytes and in the ultrastructure of the liver. One may also conclude that small size Ag NPs, which are increasingly used in human products could cause various toxigenic responses to all hepatic tissue components.

2476 Fibrinogen-Positive Ground Glass Hepatocytes in a Patient With Ulcerative Colitis

INTRODUCTION: Ground-glass hepatocyte cytoplasmic inclusions (eosinophilic, glassy cytoplasmic changes) carry a unique differential diagnosis with significant diagnostic implications. This cytoplasmic change is thought to occur from accumulation of metabolic products including glycogen, fibrinogen, or viral proteins. Few cases of patients with ground-glass, fibrinogen-positive inclusions have been observed and are thought to occur from acute states of metabolic stress. CASE DESCRIPTION/METHODS: A 64-year-old male with history of poorly controlled ulcerative colitis presented with diffuse abdominal pain, weight loss, and two months of frequent bloody bowel movements. On arrival, he was afebrile without abnormal vital signs. His appearance was non-toxic but malnourished. Abdominal exam produced diffuse tenderness but no evidence of peritonitis. Labs were notable for AST 233 U/L, ALT 486 U/L, and Alk phos 140 U/L (normal 56-119 U/L). He failed trial of medical therapy and required total abdominal colectomy. His aminotransferase levels during this time continued to worsen without explanation (peak AST 710 U/L and ALT 1145 U/L). Serum infectious disease workup including Hepatitis C antibody, HIV Antibody/Antigen screen, Hepatitis B core antibody and surface antigen, Hepatitis E IgM antibody, HSV-1 and 2 PCR, and Varicella-zoster PCR were all negative. Serum IgG, IgA, TTG-IgA, anti-smooth muscle antibody and anti-nuclear antibody testing were unremarkable. Abdominal ultrasound with doppler showed patency of the portal, splenic, and hepatic veins without thrombus. Liver biopsy showed moderately active steatohepatitis, and hepatocytes displayed ground-glass cytoplasmic appearance. PAS staining showed weak to no staining of ground-glass regions. Due to the lack of positive PAS staining, fibrinogen staining of the liver specimen was obtained, which showed diffuse positivity within hepatocytes. DISCUSSION: A rarely reported finding, fibrinogen accumulation in hepatocytes can occur from inherited disease and from acquired errors in endoplasmic reticulum processing in a setting of metabolic stress. Ground glass hepatocyte inclusions staining positive with fibrinogen have only been associated with the acquired causes in humans. Only 3 cases, to our knowledge, have shown similar findings. After 1 year follow-up from total colectomy, his aminotransferases had returned to normal range.

Hepatitis B Surface Antigen Activates Unfolded Protein Response in Forming Ground Glass Hepatocytes of Chronic Hepatitis B.

Ground glass hepatocytes (GGHs), a histological hallmark of chronic hepatitis B virus (HBV) infection, contain excessive hepatitis surface antigen (HBsAg) in the endoplasmic reticulum (ER), which is linked to unfolded protein response (UPR). The mechanism by which HBV activates UPR has not been fully defined. To investigate this, HepG2-NTCP cells and primary human hepatocytes (PHHs) were either infected with HBV or transduced with adenoviral vectors expressing replication-competent HBV genome or individual HBV genes. UPR markers were evaluated by qPCR, Western blotting, and immunofluorescence. Apoptosis and cell viability were measured by Caspase-3/7 and ATPlite assay respectively. We found that UPR markers were induced by the overexpression of HBsAg in HepG2-NTCP cells and PHHs. Elevation of UPR-induced genes showed a dose-dependent correlation with HBsAg levels. In HBV-infected livers, GGHs also demonstrated excessive accumulation of HBsAg associated with increased BIP/GRP78 staining, a marker of UPR. Prolonged activation of UPR by HBsAg overexpression induced signs of apoptosis. Overexpression of HBsAg can induce ER stress through protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway in vitro, and may be linked to the appearance of GGHs. The activation of UPR by HBsAg may sensitize hepatocytes to cell death and result in possible subsequent cellular changes leading to a premalignant phenotype.

P1‐017: Ground glass hepatocytes provide targets for prevention and therapy of HBV‐related hepatocellular carcinoma

Journal of Viral HepatitisVolume 25, Issue S2 p. 40-41 Abstracts P1-017: Ground glass hepatocytes provide targets for prevention and therapy of HBV-related hepatocellular carcinoma First published: 12 June 2018 https://doi.org/10.1111/jvh.11_12923Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume25, IssueS2Special Issue: 16th International Symposium on Viral Hepatitis and Liver Diseases (ISVHLD) held from June 14th to 17th, 2018, in Toronto, CanadaJune 2018Pages 40-41 RelatedInformation

Ground glass hepatocytes provide targets for therapy or prevention of hepatitis B virus-related hepatocellular carcinoma

Ground glass hepatocyte (GGH) represents a histologic hallmark of chronic hepatitis B virus (HBV) infection and is characterized by the accumulation of pre-S mutant surface antigens in the endoplasmic reticulum (ER). In the past decade, GGHs have been recognized as pre-neoplastic lesions of hepatocellular carcinoma (HCC). The accumulation of pre-S mutant protein in ER may induce a misfolded protein response or ER stress signals with activation of VEGF/Akt/mTOR and COX-2/NF-κB signals, leading to oxidative DNA damage, aneuploidy, and genomic instability. Molecular studies revealed clonal HBV DNA integration in type II GGHs which continue to express and secrete surface antigens, representing the sustained surface antigens in the serum after NA antiviral treatment. The persistence of GGHs in the liver after anti-viral therapy not only constitute the challenge to eliminate HBV infection but also carry the high risk to develop HCC. DNA chip and ELISA kit are designing to detect the pre-S mutants in serum. Novel or second generation anti-HBV drugs are under phase II development and include the combination of anti-virals, immunomodulators, agents for host DNA damage, and siRNA to target at the transcription of HBsAg gene in cccDNA or integrated HBV DNA. In the past years, we explored the possibility to provide drugs or natural agents targeting at ER stress signals in GGHs to prevent HCC development. In a transgenic mice model of pre-S mutant and HBx, a combination of silymarin and resveratrol targeting at mTOR and NF-κB signals could reduce a 80% of HCC development. In a pilot clinical trial, liposomal curcumin (Meriva®) combined with anti-virals and immumodulators P1101 have been designed and attempted to eliminate the serum surface antigen and hence the recurrence of HCC after surgical resection. Therefore, the detection of pre-S mutants in serum or GGHs in the liver should provide rational target design for therapy or prevention of HCC in these high risk patients of chronic HBV infection.

Incidental findings on liver autopsy with specific emphasis on Hepatitis B

Background: Silent liver disease is a major cause of morbidity and mortality in the population due to its silent progression to end stage liver disease without significant symptoms. These have to be detected at an earlier stage to reduce the morbidity. This study is aims at correlating the gross and microscopic morphology of 100 liver specimens obtained by autopsy and to correlate with the clinical parameters. Methods: 100 liver specimens obtained by the autopsy of 100 patients, who died of causes like road traffic accidents, poisoning etc. with no medical history during the period from 2007 to 2012, were taken for the study. Histological samples of liver specimens were stained with the routine heamatoxylin and eosin staining, along with special stains like PAS, Reticulin, Perl’s Prussian blue and Orcein staining. Result: The most common pathology was steatosis (25%) following normal histology (49%), which was followed by chronic venous congestion (12%), chronic hepatitis (6%), cirrhosis (4%), liver cell dysplasia (2%) and metastases (2%). These findings have been correlated with age, sex and special staining. Orcein staining identified ground-glass hepatocytes with detection of Hepatitis B surface antigen in a background of chronic hepatitis and cirrhosis, indication the most probable etiology of Hepatitis B. Conclusion: This study reiterates the unexpected higher prevalence of NASH among the younger generation and the importance of clinical autopsy in understanding the magnitude of clinically silent liver lesions, mainly Hepatitis B. DOI: 10.21276/APALM.1419

Histopathologic criteria to differentiate autoimmune hepatitis from chronic viral hepatitis (B, C) in liver biopsy

Abstract Introduction Chronic hepatitis is a common disease in the community which is caused mainly by viral agents or immune factors. Differential diagnosis of hepatitis in the treatment is very important due to different therapy. Aim This study is designed to determine importance of histological findings of autoimmune hepatitis (AIH) to distinguish from other chronic hepatitis. Material and methods In a cross-sectional study, 112 patients with hepatitis (autoimmune, B and C) were assessed about histopathological criteria on liver biopsy. Results Among the 12 indicators in histopathologic criteria, 5 indexes of interface hepatitis, lymphoid follicle in port area, emperipolesis, hepatic rosette and ground glass hepatocyte have the ability to differentiate types of hepatitis. Discussion Clinical and laboratory features of hepatitis B and C can overlap with those of AIH. From therapeutic point of view, distinguishing AIH from viral hepatitis is vital, as their treatment is totally different. Some histopathologic criteria on liver biopsy accompanied with other clinical and paraclinical findings help differentiation of them. Conclusions Histopathological parameters can differentiate types of hepatitis at an acceptable level which among them, interface hepatitis is used to differentiate between AIH and hepatitis B, lymphoid follicle in port area is used to differentiate hepatitis C and the other two types, emperipolesis to differentiate AIH from the other two types, hepatic rosette to differentiate AIH from the other two types and ground glass hepatocyte to differentiate hepatitis B from the other two types.

Resistance of ground glass hepatocytes to oral antivirals in chronic hepatitis B patients and implication for the development of hepatocellular carcinoma.

Ground glass hepatocytes (GGHs) have been shown to predict the development of hepatocellular carcinoma (HCC). Type I GGH and type II GGH harbor hepatitis B virus (HBV) pre-S1 and pre-S2 deletion mutants, respectively. Whether anti-HBV therapy can inhibit the expression of GGHs and potentially reduce HCC development is explored in this study. Two sets of liver specimens were included: the first contained 31 paired biopsy specimens obtained from chronic HBV patients receiving oral nucleos(t)ide analogue (NA) treatment; the second contained 186 resected liver tissues obtained from HBV-related HCC patients receiving surgery: 82 received NA before surgery and 104 did not. Compared with the baseline biopsy specimens, type I (P=0.527) and type II GGH (P=0.077) were not significantly decreased after 48 weeks of NA treatment in the first set of patients. In the second set, despite suppression of viral load (P<0.001) and periportal necrosis (P=0.006) in treated patients, GGH (P=0.594), cccDNA (P=0.172) and serum pre-S mutants (p=0.401) were not significantly suppressed. A significant decrease of type I (P=0.049) and type II GGH (P=0.029) could only be observed in patients after long duration of treatment (median duration: 4.3 years). In the treated patients, the persisted type II GGH remained an independent variable associated with decreased local recurrence-free survival of HCC (P=0.019) as in non-treated patients (P=0.001). In conclusion, the persistence of GGHs could explain the residual risk of HCC development under anti-HBV treatment. Therefore, intrahepatic GGHs and pre-S mutant are potential additional targets for HCC prevention in patients already receiving anti-HBV treatment.

Hepatitis B virus PreS2-mutant large surface antigen activates store-operated calcium entry and promotes chromosome instability.

Hepatitis B virus (HBV) is a driver of hepatocellular carcinoma, and two viral products, X and large surface antigen (LHBS), are viral oncoproteins. During chronic viral infection, immune-escape mutants on the preS2 region of LHBS (preS2-LHBS) are gain-of-function mutations that are linked to preneoplastic ground glass hepatocytes (GGHs) and early disease onset of hepatocellular carcinoma. Here, we show that preS2-LHBS provoked calcium release from the endoplasmic reticulum (ER) and triggered stored-operated calcium entry (SOCE). The activation of SOCE increased ER and plasma membrane (PM) connections, which was linked by ER- resident stromal interaction molecule-1 (STIM1) protein and PM-resident calcium release- activated calcium modulator 1 (Orai1). Persistent activation of SOCE induced centrosome overduplication, aberrant multipolar division, chromosome aneuploidy, anchorage-independent growth, and xenograft tumorigenesis in hepatocytes expressing preS2- LHBS. Chemical inhibitions of SOCE machinery and silencing of STIM1 significantly reduced centrosome numbers, multipolar division, and xenograft tumorigenesis induced by preS2-LHBS. These results provide the first mechanistic link between calcium homeostasis and chromosome instability in hepatocytes carrying preS2-LHBS. Therefore, persistent activation of SOCE represents a novel pathological mechanism in HBV-mediated hepatocarcinogenesis.

Hepatitis B virus pre-S2 mutant large surface protein inhibits DNA double-strand break repair and leads to genome instability in hepatocarcinogenesis.

Although hepatitis B virus (HBV) has been established to cause hepatocellular carcinoma (HCC), the exact mechanism remains to be clarified. Type II ground glass hepatocytes (GGHs) harbouring the HBV pre-S2 mutant large surface protein (LHBS) have been recognized as a morphologically distinct hallmark of HCC in the advanced stages of chronic HBV infection. Considering its preneoplastic nature, we hypothesized that type II GGH may exhibit high genomic instability, which is important for the carcinogenic process in chronic HBV carriers. In this study we found that pre-S2 mutant LHBS directly interacted with importin α1, the key factor that recognizes cargos undergoing nuclear transportation mediated by the importin α/β-associated nuclear pore complex (NPC). By interacting with importin α1, which inhibits its function as an NPC factor, pre-S2 mutant LHBS blocked nuclear transport of an essential DNA repair and recombination factor, Nijmegen breakage syndrome 1 (NBS1), upon DNA damage, thereby delaying the formation of nuclear foci at the sites of DNA double-strand breaks (DSBs). Pre-S2 mutant LHBS was also found to block NBS1-mediated homologous recombination repair and induce multi-nucleation of cells. In addition, pre-S2 mutant LHBS transgenic mice showed genomic instability, indicated by increased global gene copy number variations (CNVs), which were significantly higher than those in hepatitis B virus X mice, indicating that pre-S2 mutant LHBS is the major viral oncoprotein inducing genomic instability in HBV-infected hepatocytes. Consistently, the human type II GGHs in HCC patients exhibited increased DNA DSBs representing significant genomic instability. In conclusion, type II GGHs harbouring HBV pre-S2 mutant oncoprotein represent a high-risk marker for the loss of genome integrity in chronic HBV carriers and explain the complex chromosome changes in HCCs. Mouse array CGH raw data: GEO Accession No. GSE61378 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE61378).

HBsAg expression of liver correlates with histological activities and viral replication in chronic hepatitis B

Introduction. The intrahepatic hepatitis B surface antigens (HBsAg) expression is related to disease progression of chronic hepatitis B. We examined the features of intrahepatic HBsAg expression.Material and methods. A total of 181 patients with e antigen positive chronic hepatitis B were enrolled. Patterns and semi-quantitative measurement of intrahepatic HBsAg expression were analyzed. The association of intra-hepatic hepatitis HBsAg expression with clinical, viral, and histological characteristics was evaluated.Results. Higher necroinflammation grade and greater fibrosis stage accompanied with lower serum HBV DNA and HBsAg levels were observed in patients with type II ground glass hepatocytes and 2+/3+ scales of intrahepatic HBsAg expression. Basal core promoter T1762/A1764 mutations were strongly associated with the pattern of type II ground glass hepatocytes expression (P < 0.001) and higher level of HBsAg expression (9.3 ± 8.0% vs. 4.3 ± 5.0%, P = 0.008). In multivariate analysis, basal core promoter mutations (Odds ratio: 6.356, 95% confidence interval: 1.204 ~ 33.356, P = 0.029) was associated with 2+/3+ scale of HBsAg expression.Conclusion. Both pattern and levels of intrahepatic HBsAg expression were associated with severity of liver disease in e antigen positive chronic hepatitis B. Strong relationship between intrahepatic HBsAg expression and basal core promoter 1762/A1764 mutations indicated that HBsAg expression may be the histological manifestation of hepatitis B virus genomic evolution under host immune surveillance.

The emerging role of hepatitis B virus pre-S2 deletion mutant proteins in HBV tumorigenesis.

Chronic hepatitis B virus (HBV) infection can cause hepatocellular carcinoma (HCC). Several hypotheses have been proposed to explain the mechanisms of HBV tumorigenesis, including inflammation and liver regeneration associated with cytotoxic immune injuries and transcriptional activators of mutant HBV gene products. The mutant viral oncoprotein-driven tumorigenesis is prevailed at the advanced stage or anti-HBe-positive phase of chronic HBV infection. Besides HBx, the pre-S2 (deletion) mutant protein represents a newly recognized oncoprotein that is accumulated in the endoplasmic reticulum (ER) and manifests as type II ground glass hepatocytes (GGH). The retention of pre-S2 mutant protein in ER can induce ER stress and initiate an ER stress-dependent VEGF/Akt/mTOR and NFκB/COX-2 signal pathway. Additionally, the pre-S2 mutant large surface protein can induce an ER stress-independent pathway to transactivate JAB-1/p27/RB/cyclin A,D pathway, leading to growth advantage of type II GGH. The pre-S2 mutant protein-induced ER stress can also cause DNA damage, centrosome overduplication, and genomic instability. In 5-10% of type II GGHs, there is co-expression of pre-S2 mutant protein and HBx antigen which exhibited enhanced oncogenic effects in transgenic mice. The mTOR signal cascade is consistently activated throughout the course of pre-S2 mutant transgenic livers and in human HCC tissues, leading to metabolic disorders and HCC tumorigenesis. Clinically, the presence of pre-S2 deletion mutants in sera frequently develop resistance to nucleoside analogues anti-virals and predict HCC development. The pre-S2 deletion mutants and type II GGHs therefore represent novel biomarkers of HBV-related HCCs. A versatile DNA array chip has been developed to detect pre-S2 mutants in serum. Overall, the presence of pre-S2 mutants in serum has implications for anti-viral treatment and can predict HCC development. Targeting at pre-S2 mutant protein-induced, ER stress-dependent, mTOR signal cascade and metabolic disorders may offer potential strategy for chemoprevention or therapy in high risk chronic HBV carriers.

Ground glass hepatocytes coexpressing hepatitis B virus X protein and surface antigens effect on oncogenic effects and tumorigenesis.

e15115 Background: Hepatitis B virus (HBV) X protein (HBx) and pre-S2 deletion mutant large surface antigens have been recognized as oncoproteins that induce hepatocellular carcinoma (HCC). The int...

Ground-glass hepatocytes co-expressing hepatitis B virus X protein and surface antigens exhibit enhanced oncogenic effects and tumorigenesis.

Hepatitis B virus (HBV) X protein (HBx) and pre-S2 deletion mutant large surface antigens are oncoproteins that induce hepatocellular carcinoma (HCC). The interaction of these two oncoproteins in hepatocytes and its significance in tumorigenesis remain to be elucidated. In this study, we observed the co-expression of HBx with surface antigens in ground-glass hepatocytes in 5 of 20 hepatitis B surface antigen-positive livers. In vitro, hepatocytes co-expressing HBx and a pre-S2 mutant showed enhanced expression of vascular endothelial growth factor-A, phosphorylated Akt 1/2/3, phosphorylated extracellular signal-regulated kinase 1/2, and phosphorylated mammalian target of rapamycin signals. Transgenic mice harboring both HBx and pre-S2 mutant construct plasmids developed HCCs at an average of 15.1 months, earlier than animals carrying either HBx (16.9 months) or pre-S2 mutant (24.5 months) alone. The oncogenic signals of vascular endothelial growth factor-A, phosphorylated Akt 1/2/3, phosphorylated extracellular signal-regulated kinase 1/2, and phosphorylated mammalian target of rapamycin were sequentially and differentially activated at different stages in tumorigenesis. Phosphorylated mTOR was consistently activated in transgenic and human HCCs. We conclude that ground-glass hepatocytes co-expressing HBx and surface antigens exhibit enhanced oncogenic effects and tumorigenesis in chronic HBV infections. The mTOR signal cascade may be the key regulator in HBV tumorigenesis and may be useful targets in the design of HCC therapy.