Although chronic hepatitis B virus (HBV) infection has been well-established to cause hepatocellular carcinoma (HCC), the preneoplastic lesions in liver and the exact molecular mechanism of HBV tumor genesis remains to be elucidated. Current views of HBV tumor genesis include inflammation and liver regeneration associated with cytotoxic immune injuries at the early stage, the random dysregulation of cellular growth control genes by genetic abnormalities, and the transcriptional activators of mutant HBV gene products at the advanced stage of chronic HBV infection. Besides HBx, the pre-S2 (deletion) mutant protein represents a newly recognized viral oncoprotein that is accumulated in the endoplasmic reticulum (ER) and manifests as type II ground glass hepatocytes (GGH) due to ER proliferation. The retention of pre-S2 mutant protein in GGHs can induce ER stress-dependent and –independent pathway to initiate VEGF/Akt/mTOR, NFkB/COX-2 and JAB-1/p27/RB/cyclin A, D pathway, leading to growth advantage of type II GGH. The pre-S2 mutant protein-induced ER stress response can also cause DNA damage, centrosome overduplication, and genomic instability. Type II GGHs can co-express pre-S2 mutant protein and HBx antigen which together exhibit an enhanced oncogenic effect in transgenic mice. Distinct from type II GGHs, type I GGHs usually distribute sporadically and express stronger ER stress which will lead to apoptosis. Clinically, the presence of pre-S2 deletion mutants in sera frequently develops resistance to nucleoside analogues anti-virals and closely associate with HCC development. Whether type II GGHs represent a lesion of focal adenomatous hyperplasia and directly contribute to HCC development remains to be clarified. A versatile DNA array chip and ELISA kit have been developed to detect pre-S2 mutants in serum. Overall, the current evidence suggest that the presence of GGHs, particularly type II, and the detection of pre-S2 mutants in serum has implications for anti-viral treatment and can predict HCC development in patients with chronic HBV infection. A well-designed cohort study and clinical trial should be conducted to confirm that type II GGHs represent precursor lesions of HBV-related HCCs.

Su IJ, Wu HC, Tsai HW, Wang LHC, Hsieh WC, et al. (2014) The Emerging Role of Ground Glass Hepatocytes and Hepatitis B Virus Pre-S Mutants in Anti-Viral Therapy and HBV Tumor Genesis. Ann Clin Pathol 2(6): 1041.

•    Ground glass hepatocytes
•    Pre-S mutants
•    Endoplasmic reticulum stress
•    Chronic HBV infection
•    Hepatocellular carcinoma

Although chronic Hepatitis B virus ( HBV ) has been well established to cause hepatocellular carcinoma ( HCCs ) [1], the exact mechanism leading to HBV tumorigenesis remains to be elucidated. The prevailing views of HBV tumorigenesis include inflammation and liver regeneration associated with cytotoxic immune ( CTL ) injuries, HBV DNA insertional mutagenesis, and viral oncoproteins-driven tumorigenesis [2-4]. HCC can occur at the early stage of chronic HBV infection which has active HBV replication and CTL response [3,5], while the majority of HCC cases occur at the advanced stage or anti-HBe-positive phase at the sixth decade of age [4]. In the advanced or anti-HBe-positive phase, it is believed that the mutant viral oncoproteins which can escape CTL attack play a major or driving role in the subsequent HCC development.

Hadziyannis and Popper first recognized the HBV surface antigens in the “ground glass” hepatocytes (GGH) of chronic HBV carriers [6,7]. Under electron microscopy, GGHs are characterized by an abundance of endoplasmic reticulum (ER), among which particles of surface antigens accumulate [8]. It is believed that the overloaded ER makes the cytoplasms of GGHs become “foggy” or “glassy”. Chisari, et al were the first group to demonstrate the formation of GGHs in transgenic mice which express large amount of wild type large surface antigen [9]. Two types of GGHs were later designated as type I and II GGHs [10]. Type I GGHs are usually scattered singly in the hepatic lobules with the expression of “inclusion-like” pattern of surface antigens (Figure 1A). This type of GGHs usually occurs at the early stage or in patients with active replicative diseases [11-13]. Type II GGHs, however, express a unique expression pattern of surface antigens at the cell margin (Figure 1A). Most importantly, type II GGHs consistently cluster in nodules and usually occur at the advanced stages or anti-HBe-positive phase [14], and are frequently associated with cirrhosis or hepatocellular carcinoma (HCC). The consistent clustering distribution of type II GGHs, especially in the non-tumorous liver tissues of patients with HCC receiving surgery, drives us to hypothesize that type II GGHs may represent clonal preneoplastic or focal adenomatous lesions of HCC [15]. GGHs have been proposed to progress from focal adenomatous hyperplasia, adenoma, and HCC in transgenic mice harboring wild type HBV genome [16]. The exact pathogenesis and molecular mechanism of GGHs in HBV tumor genesis, however, remains uncharacterized, although chronic liver injury, necroinflammation, and regenerative hyperplasia have been proposed to explain the disease progression in different transgenic mice model [17].

In the past decade, we studied in detail the biologic and molecular significance of type II GGHs which contain a unique form of pre-S2 deletion mutant protein. The significance of pre-S2 mutant proteins in HCC development, the signal pathway initiated by pre-S2 mutant proteins, the clinical significance of pre-S mutants in serum, and the transgenic mice model of pre-S2 mutant tumorigenesis were studied. Importantly, we developed a combined DNA array chip to detect pre-S2 mutant gene and an ELISA kit to detect pre-S2 mutant protein in serum as the predictive marker of HCC in chronic HBV carriers.

Ground glass hepatocytes contain pre-S deletion mutant proteins which accumulate in endoplasmic reticulum (ER) and induce ER stress signals

By dissecting the cirrhotic nodules in liver tissues of chronic HBV hepatitis patients, we are surprised to find that type II GGHs consistently harbored pre-S2 mutants with in-frame deletions over the pre-S2 regions (predominantly, nt. 2–55 or 4–57) with or without point mutations at the start codon (ATG–ATA) of the S promoter. These mutations lead to a decreased synthesis of middle and small surface antigens and result in a defective secretion of the mutant large surface antigens which then accumulate in ER, leading to the GGH formation in chronic HBV infection [10,15]. The deletion site at the pre-S2 region coincides with the epitope of cytotoxic T-cell and B-cell neutralization responses and suggests that the pre-S2 deletion mutants represent an immune escape mutant [18,19]. In cocnsistence with the immune escape epitope of the pre-S2 deletion site, the liver tissues containing type II GGHs are usually devoid of inflammatory infiltrate. Distinct from type II GGHs, the singly distributed type I GGHs contain entirely different pre-S mutants with variable deletions over the pre-S1 regions. The liver lobules containing the type I GGHs usually show presence of inflammatory infiltrate, distinct from that in type II GGHs. The deletion sites at the pre-S1 region may interfere with the transcriptional activities of the S promoter and affect the regulation of HBV replication and synthesis of small surface antigens [20,21]. The pre-S1 containing large surface antigen (LHB) exhibits a dual topology, and only half of the LHB translocate post-translationally into the ER lumen [22,23]. The pre-S regions that remain in cytoplasmic orientation can bind to the cytosolic heat shock protein Hsc70 [24] and presumably interact with the core particle during virion assembly [25]. In HCC patients, the pre-S mutants are prevalent for up to 63%, including mutants with combinations of deletions over the pre-S1 and pre-S2 regions (Figure 1B) [26,27].

The accumulation of mutant or unfolded proteins causes stress in ER that is sensed by the glucose-regulated protein 78 (Grp78). Unfolded proteins will sequester GRP78 and dissociate from three ER transmembrane transducers leading to their activation [28]. It has been demonstrated that the secretion of surface proteins was compromised by pre-S deletions, especially preS2 mutants [10]. In addition, ectopic expression of pre-S mutant proteins in Huh-7 cells increased the levels of ER chaperones (Grp78 and 94) and activated PERK and C-jun N-terminal kinase (JNK) [10]. Northern and Western blot analyses revealed that the pre-S1 mutant induced stronger levels of ER chaperone (Grp78 and 94) response, calcium release, cyclooxygenase-2 (COX-2) and inflammatory cytokines, and oxidative stress intermediates, which tend to result in apoptosis [10,29,30]. The pre-S2 mutants, albeit inducing a weaker level of ER stress signal, exhibited higher levels of mutation frequency and transforming capabilities in primary hepatocyte cell line HH4 [31].

ER stress-induced by pre-S2 mutants leads to oxidative DNA damage, genomic instability, and transforming capabilities in transgenic mice model

The induction of ER stress has been shown to increase the level of reactive oxygen species (ROS), NFκB activation and cyclooxygenase-2 (COX-2) expression and thereby induces oxidative DNA damages (Figure 2) [28,29,32]. Treatments with antioxidants reduced ER stress and improved protein folding [33]. As expected, the expression of pre-S mutant proteins induced oxidative DNA damages, as demonstrated by the increase in 8-hydroxyguanonine on the DNA lesion and increased levels of 8-oxoguanine glycosylase 1 (ogg1) and X-ray cross-complementation 1 (xrcc1) [30]. These studies suggest the presence of genomic instability in GGHs [34]. Notably, as a promising gene transactivator and an ER stress inducer, pre-S2 mutant protein also promotes centrosome instability through two independent mechanisms. First, pre-S2 mutant protein could upregulate cyclin A and sustained cyclin D1 and cyclin-dependent kinase-4 via gene transactivation. This event subsequently promoted cell cycle progression even in the presence of ER stress and resulted in nodular proliferation in transgenic mice livers [31]. Second, ER stress facilitates the release of calcium from the ER and thereby activates calciumdependent calpain proteases. Notably, cyclin A is a substrate of calpain and the proteolysis results in cytoplasmic redistribution of cyclin A and thereby stimulates centrosome overduplication [35]. These studies demonstrate that pre-S2 mutant protein is a direct driver of genomic instability through the induction of DNA damages and centrosome abnormality.

The most important molecular mechanism initiated by pre-S2 mutant is the VEGF/Akt/mTOR signal pathway which is activated at the early, middle, and advanced stages of transgenic livers harboring pre-S2 deletion mutant (Figure 2) [36,37]. The mTOR signaling is commonly activated in human HCC tissues and represents a candidate target for therapy. The transforming ability of pre-S2 mutant proteins has been investigated in an immortalized human hepatocyte line HH4 which is immortalized by human papilloma virus [31]. In addition, the transgenic mice carrying pre-S2 mutant developed HCC [38]. These studies further support the role of pre-S2 mutants and GGHs in HBV hepatocarcinogenesis [14,39].

Aside from the ER stress-dependent signaling pathways, a distinct ER stress-independent response has been found specifically for pre-S2 deletion mutant protein and is significant for their biologic and carcinogenic preferences [10]. The pre-S2 mutant protein specifically interacts with c-Jun activation domain binding protein 1 (JAB1), which enhances activator protein-1 transcriptional activity and cell proliferation [34]. Through its binding to JAB1, the pre-S2 mutant protein induces JAB1 nuclear translocation, which activates p27/retinoblastoma/Cdk2/cyclin A pathways and leads to cell cycle progression and centrosome over-duplication [31,35]. These findings have provided clear mechanisms for the growth advantage induced by the pre-S2 mutant protein.

Two HBV viral proteins, the X protein (HBx) and pre-S2 deletion mutant protein, have been considered to have either direct or indirect effects in HBV hepatocarcinogenesis [40]. Other than the molecular mechanisms of pre-S2 deletion mutants in hepatocarcinogenesis as mentioned above, HBx also has been shown to mediate the activation of multiple signal pathways including the mammalian target of rapamycin (mTOR) signal cascade [41-45]. In our laboratory, we observed a strong HBx expression in the cytosolic fraction of GGHs in 5 of 20 HBsAgpositive human livers. Interestingly, HBx was consistently coexpressed with HBsAg, but not vice versa, in GGHs. The expression of both oncoproteins, however, was only rarely detected in HCC tissues [37]. Transgenic mice harboring the HBx, pre-S2 mutant, and a double (HBx plus pre-S2 mutant) have been established in our laboratory. We observed that the transgenic livers harboring double construct plasmids developed HCCs at an average of 15.1 months, earlier than that of HBx (16.9 months) or pre-S2 mutant (24.5 months) alone. The exact role of HBx and pre-S2 mutant protein, either alone or in combination, in human HCC development remains to be clarified.

Based on our studies cited above, we propose that GGHs, especially type II which harbored the pre-S2 deletion mutants, may represent the precursor lesions of HCC in the advanced stage of chronic HBV infection. As mentioned above, the mechanism of HBV tumorigenesis is complicated and several factors may cooperate to jointly promote the tumorigenesis. In patients who developed HCC at early stage of life, chronic liver injuries and subsequent liver regeneration may play an important role as inferred from the transgenic animal models, mainly from the group of Chisari, et al [9,17]. Distinct from the concept that type II GGHs plays a direct role for HBV tumorigenesis, they proposed that GGHs overexpressing large surface antigens will undergo cell death and the regenerative hyperplasia of other undefined hepatocytes may progress inexorably and indirectly contribute to HBV tumorigenesis [16,46]. Whether chronic liver injury is mandatory for HCC development is still controversial [47]. In the human liver tissues in which type II GGHs located, there is usually no inflammatory infiltrate observed, distinct from that in type I GGHs or liver tissues of chronic HBV exacerbation [14,26]. Since pre-S2 deletion mutant is an immune escape variant due to the deletion of B and T cell epitope [18,19], the liver tissues harboring type II GGHs can be devoid of chronic necroinflammatory change. Therefore, the mechanism of tumorigenesis exhibited by type II GGHs in human may be different from that of GGHs proposed by Chisari, et al in transgenic mice which harbor wild type large surface antigen.

Presence of pre-S2 mutants in serum predicts the resistance of nucleoside analogues anti-virals and a higher risk of HCC development.

The emergence of pre-S deletion mutants occurs during the natural course of HBV infection, possibly due to selective pressure by the immune system [48,49]. The frequencies of pre-S mutation increased successively in different stage of chronic HBV infection. In a meta-analysis study [50], the summarized results showed that pre-S mutation was detected in around 10% of asymptomatic HBsAg carriers, 20% of patients with chronic hepatitis B, 35% of patients with liver cirrhosis and 50% patients with HCC. The pre-S2 deletion mutants are more frequently detected in anti-HBeAg-positive patients and in patients with HCC than in HBeAg-positive patients [27,48,51]. The ratio of pre-S mutant clones related to wild type in serum also accumulates, as it was 6.4% at high replicative phase, 13% at intermediate, and 37.5% at low or nonreplicative phases [27]. Therefore, pre-S2 mutants represent a significant proportion of virus in advanced stage patients [52,53].

Although anti-viral nucleoside analogues therapy has been associated with a lower risk of HCC development or recurrence after liver resection in chronic HBV carriers [54], the frequencies of pre-S mutation have been reported to be increased after antiviral therapy by nucleos(t)ide analogues which is closely associated with the drug resistance and predict the high risk development of HCC [55,56]. Interestingly, in the control group treated with alpha-interferon, the pre-S2 mutants were significantly reduced or absent, suggesting that alpha-interferon may degrade or inhibit the synthesis of pre-S2 mutant proteins [55]. The presence of pre-S mutants, especially pre-S2 mutant, has been found to be significantly associated with the risk of HCC development [55-60]. Pre-S deletion mutants detected in serum has also been reported to increase the risk of post-operative recurrence of HCC [61]. Of particular note, pre-S mutation could occur early in age and significantly associated with HCC in pediatric patients [62,63]. Pre-S2 deletion in sera can be detected in nearly half of children with HCC, in contrast to none in children with chronic HBV infection [62]. By using tissue samples, pre-S2 deletion mutants can be detected in about 80% of pediatric HCC [63]. Overall, the combined effects of cell cycle progression, genomic instabilities, and survival advantage exhibited by pre-S2 mutant proteins strongly suggest that type II GGHs are potential preneoplastic loci for HCC development and de novo recurrence after surgical resection. In a cohort of 82 patients with HBV-related HCC who received curative surgery [64], type II GGHs were found to be the independent variables associated with late recurrence and the overall survival. In the transgenic mice model using whole HBV genome, Chisari’s group demonstrated a sequential evolution from GGHs to focal hyperplasia, adenomatous hyperplasia or dysplasia, and finally the development of HCC [16]. Therefore, they hypothesized that GGHs represent the precursor lesions of human HCC development.

Development of DNA Chip and ELISA kit to detect pre-S2 mutants in serum as the predictive hallmark of HCC

To efficiently detect pre-S deletion mutants in serum, we have successfully developed the oligonucleotide Pre-S Gene Chip and the ELISA system to detect the pre-S deletion mutants in sera. The Pre-S Gene Chip contains 42 DNA probes that target the pre-S region of the LHBS gene, offering a highly sensitive and specific method for pre-S deletion detection with short turnaround time (< 3 days) [65]. Screening the pre-S deletion mutants revealed interesting findings that the detection rate of pre-S mutants were relatively low (7%) in the sera of patients with acute exacerbation of chronic HBV infection but gradually increased in later periods of chronic HBV infection, as they were 37% in advanced stage of chronic HBV carriers, and as high as 60% in HCC patients [65]. Combined detection of pre-S mutants and other markers of HBV replication such as HBeAg and viral loads is believed to offer a reliable predictive method for predicting HCC risks in chronic HBV carriers.

In this review, we provide an overview to provide evidence on the emerging role of HBV pre-S2 deletion mutant protein in HBV tumorigenesis, although cohort studies and genetic studies are needed to clarify whether type II GGHs represent the preneoplastic or adenomatous lesions and directly contribute to HCC development. The HBV pre-S2 deletion mutant proteins are retained in the ER and induce ER stress response. Series of ER stress-dependent and -independent growth signals are then activated. Among the diverse pathways, mTOR-mediated signal cascade represent a major mechanism for the disturbed metabolism, genomic instability, and growth advantage, which can potentially drive the type II GGHs toward the pre-neoplastic and neoplastic lesions. To identify the patients at high risk for HCC development represents the major task in combating chronic HBV infection in the coming decades. The development of a DNA chip and ELISA kit for detecting pre-S2 deletion mutant will meet this demand. Chemopreventive or therapeutic agents can then be provided to these high risk HBV carriers to prevent from HCC development.

This study was supported by grants from National Health Research Institutes and National Science Council, Taiwan (Dr. Su and Dr. Huang).

1. Beasley RP, Hwang LY, Lin CC, Chien CS. Hepatocellular carcinoma and hepatitis B virus. A prospective study of 22 707 men in Taiwan. See comment in PubMed Commons below Lancet. 1981; 2: 1129-1133.

2. Su IJ, Hsieh WC, Tsai HW2, Wu HC1. Chemoprevention and novel therapy for hepatocellular carcinoma associated with chronic hepatitis B virus infection. See comment in PubMed Commons below Hepatobiliary Surg Nutr. 2013; 2: 37-39.

3. Sitia G, Aiolfi R, Di Lucia P, Mainetti M, Fiocchi A, Mingozzi F. Antiplatelet therapy prevents hepatocellular carcinoma and improves survival in a mouse model of chronic hepatitis B. See comment in  PubMed Commons below Proc Natl Acad Sci U S A. 2012; 109: E2165- 2172.

4. Liaw YF, Chu CM. Hepatitis B virus infection. See comment in PubMed Commons below Lancet. 2009; 373: 582-592.

5. Chisari FV, Klopchin K, Moriyama T, Pasquinelli C, Dunsford HA, Sell S. Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice. See comment in PubMed Commons below Cell. 1989; 59: 1145-1156.

6. Hadziyannis S, Gerber MA, Vissoulis C, Popper H. Cytoplasmic hepatitis B antigen in “ground-glass” hepatocytes of carriers. See comment in PubMed Commons below Arch Pathol. 1973; 96: 327-330.

7. Popper H. The ground glass hepatocyte as a diagnostic hint. See comment in PubMed Commons below Hum Pathol. 1975; 6: 517-520.

8. Shikata T. Australia antigen in liver tissue--an immunofluorescent and immunoelectron microscopic study. See comment in PubMed Commons below Jpn J Exp Med. 1973; 43: 231-245.

9. Chisari FV, Pinkert CA, Milich DR, Filippi P, McLachlan A, Palmiter RD. A transgenic mouse model of the chronic hepatitis B surface antigen carrier state. See comment in PubMed Commons below Science. 1985; 230: 1157-1160.

10. Wang HC, Wu HC, Chen CF, Fausto N, Lei HY, Su IJ. Different types of ground glass hepatocytes in chronic hepatitis B virus infection contain specific pre-S mutants that may induce endoplasmic reticulum stress. Am J Pathol 2003; 163: 2441-2449.

11. Gudat F, Bianchi L, Sonnabend W, Thiel G, Aenishaenslin W, Stalder GA. Pattern of core and surface expression in liver tissue reflects state of specific immune response in hepatitis B. See comment in PubMed Commons below Lab Invest. 1975; 32: 1-9.

12. Su IJ, Kuo TT, Liaw YF. Hepatocyte hepatitis B surface antigen. Diagnostic evaluation of patients with clinically acute hepatitis B surface antigen-positive hepatitis. See comment in PubMed Commons below Arch Pathol Lab Med. 1985; 109: 400-402.

13. Hsu HC, Lin YH, Chang MH, Su IJ, Chen DS. Pathology of chronic hepatitis B virus infection in children: with special reference to the intrahepatic expression of hepatitis B virus antigens. See comment in PubMed Commons below Hepatology. 1988; 8: 378-382.

14. Su IJ, Lai MY, Hsu HC, Chen DS, Yang PM, Chuang SM. Diverse virological, histopathological and prognostic implications of seroconversion from hepatitis B e antigen to anti-HBe in chronic hepatitis B virus infection. See comment in PubMed Commons below J Hepatol. 1986; 3: 182-189.

15. Fan YF, Lu CC, Chang YC, Chang TT, Lin PW, Lei HY, et al. Identification of a pre-S2 mutant in hepatocytes expressing a novel marginal pattern of surface antigen in advanced diseases of chronic hepatitis B virus infection. J Gastroenterol Hepatol 2000; 15: 519-528.

16. Toshkov I, Chisari FV, Bannasch P. Hepatic preneoplasia in hepatitis B virus transgenic mice. See comment in PubMed Commons below Hepatology. 1994; 20: 1162-1172.

17. Chisari FV1. Hepatitis B virus transgenic mice: insights into the virus and the disease. See comment in PubMed Commons below Hepatology. 1995; 22: 1316-1325.

18. Tai PC, Banik D, Lin GI, Pai S, Pai K, Lin MH. Novel and frequent mutations of hepatitis B virus coincide with a major histocompatibility complex class I-restricted T-cell epitope of the surface antigen. See comment in PubMed Commons below J Virol. 1997; 71: 4852-4856.

19. Tai PC, Suk FM, Gerlich WH, Neurath AR, Shih C. Hypermodification and immune escape of an internally deleted middle-envelope (M) protein of frequent and predominant hepatitis B virus variants. See comment in PubMed Commons below Virology. 2002; 292: 44-58.

20. Melegari M, Scaglioni PP, Wands JR. The small envelope protein is required for secretion of a naturally occurring hepatitis B virus mutant with pre-S1 deleted. See comment in PubMed Commons below J Virol. 1997; 71: 5449-5454.

21. Xu Z, Yen TS. Intracellular retention of surface protein by a hepatitis B virus mutant that releases virion particles. See comment in PubMed Commons below J Virol. 1996; 70: 133-140.

22. Bruss V, Lu X, Thomssen R, Gerlich WH. Post-translational alterations in transmembrane topology of the hepatitis B virus large envelope protein. See comment in PubMed Commons below EMBO J. 1994; 13: 2273-2279.

23. Ostapchuk P, Hearing P, Ganem D. A dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis B viral morphogenesis. See comment in PubMed Commons below EMBO J. 1994; 13: 1048-1057.

24. Loffler-Mary H, Werr M, Prange R. Sequence-specific repression of cotranslational translocation of the hepatitis B virus envelope proteins coincides with binding of heat shock protein Hsc70. Virology 1997; 235: 144-152.

25. Le Pogam S, Shih C. Influence of a putative intermolecular interaction between core and the pre-S1 domain of the large envelope protein on hepatitis B virus secretion. See comment in PubMed Commons below J Virol. 2002; 76: 6510-6517.

26. Su IJ, Wang HC, Wu HC, Huang WY. Ground glass hepatocytes contain pre-S mutants and represent preneoplastic lesions in chronic hepatitis B virus infection. J Gastroenterol Hepatol 2008; 23: 1169-1174.

27. Fan YF, Lu CC, Chen WC, Yao WJ, Wang HC, Chang TT. Prevalence and significance of hepatitis B virus (HBV) pre-S mutants in serum and liver at different replicative stages of chronic HBV infection. See comment in PubMed Commons below Hepatology. 2001; 33: 277-286.

28. Malhi H, Kaufman RJ. Endoplasmic reticulum stress in liver disease. See comment in PubMed Commons below J Hepatol. 2011; 54: 795- 809.

29. Hung JH, Su IJ, Lei HY, Wang HC, Lin WC, Chang WT. Endoplasmic reticulum stress stimulates the expression of cyclooxygenase-2 through activation of NF-kappaB and pp38 mitogen-activated protein kinase. See comment in PubMed Commons below J Biol Chem. 2004; 279: 46384-46392.

30. Hsieh YH, Su IJ, Wang HC, Chang WW, Lei HY, Lai MD. Pre-S mutant surface antigens in chronic hepatitis B virus infection induce oxidative stress and DNA damage. See comment in PubMed Commons below Carcinogenesis. 2004; 25: 2023-2032.

31. Wang HC, Chang WT, Chang WW, Wu HC, Huang W, Lei HY. Hepatitis B virus pre-S2 mutant upregulates cyclin A expression and induces nodular proliferation of hepatocytes. See comment in PubMed Commons below Hepatology. 2005; 41: 761-770.

32. Wang S, Kaufman RJ. The impact of the unfolded protein response on human disease. See comment in PubMed Commons below J Cell Biol. 2012; 197: 857-867.

33. Malhotra JD, Miao H, Zhang K, Wolfson A, Pennathur S, Pipe SW. Antioxidants reduce endoplasmic reticulum stress and improve protein secretion. See comment in PubMed Commons below Proc Natl Acad Sci U S A. 2008; 105: 18525-18530.

34. Hsieh YH, Hsu JL, Su IJ, Huang W. Genomic instability caused by hepatitis B virus: into the hepatoma inferno. See comment in PubMed Commons below Front Biosci (Landmark Ed). 2011; 16: 2586-2597.

35. Wang LH, Huang W, Lai MD, Su IJ. Aberrant cyclin A expression and centrosome overduplication induced by hepatitis B virus pre-S2 mutants and its implication in hepatocarcinogenesis. See comment in PubMed Commons below Carcinogenesis. 2012; 33: 466-472.

36. Yang JC, Teng CF, Wu HC, Tsai HW, Chuang HC, Tsai TF. Enhanced expression of vascular endothelial growth factor-A in ground glass hepatocytes and its implication in hepatitis B virus hepatocarcinogenesis. See comment in PubMed Commons below Hepatology. 2009; 49: 1962-1971.

37. Wu HC, Tsai HW, Teng CF, Hsieh WC, Lin YJ, Wang LHC, et al. Groundglass hepatocytes co-expressing hepatitis B virus X protein and surface antigens exhibit enhanced oncogenic effects and tumorigenesis. Human Pathology. 2014; 45: 1294-1301.

38. Wang HC, Huang W, Lai MD, Su IJ. Hepatitis B virus pre-S mutants, endoplasmic reticulum stress and hepatocarcinogenesis. See comment in PubMed Commons below Cancer Sci. 2006; 97: 683-688.

39. Mathai AM, Alexander J, Kuo FY, Torbenson M, Swanson PE, Yeh MM. Type II ground-glass hepatocytes as a marker of hepatocellular carcinoma in chronic hepatitis B. See comment in PubMed Commons below Hum Pathol. 2013; 44: 1665-1671.

40. Tan A, Yeh SH, Liu CJ, Cheung C, Chen PJ. Viral hepatocarcinogenesis: from infection to cancer. See comment in PubMed Commons below Liver Int. 2008; 28: 175-188.

41. Benn J, Schneider RJ. Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade. See comment in PubMed Commons below Proc Natl Acad Sci U S A. 1994; 91: 10350-10354.

42. Chan CF, Yau TO, Jin DY, Wong CM, Fan ST, Ng IO. Evaluation of nuclear factor-kappaB, urokinase-type plasminogen activator, and HBx and their clinicopathological significance in hepatocellular carcinoma. Clin Cancer Res. 2004; 10: 4140-4149.

43. Lee YI, Kang-Park S, Do SI, Lee YI. The hepatitis B virus-X protein activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade. See comment in PubMed Commons below J Biol Chem. 2001; 276: 16969-16977.

44. Lee YH, Yun Y. HBx protein of hepatitis B virus activates Jak1-STAT signaling. See comment in PubMed Commons below J Biol Chem. 1998; 273: 25510-25515.

45. Yen CJ, Lin YJ, Yen CS, Tsai HW, Tsai TF, Chang KY. Hepatitis B virus X protein upregulates mTOR signaling through IKKβ to increase cell proliferation and VEGF production in hepatocellular carcinoma. See comment in PubMed Commons below PLoS One. 2012; 7: e41931.

46. Dunsford HA, Sell S, Chisari FV. Hepatocarcinogenesis due to chronic liver cell injury in hepatitis B virus transgenic mice. See comment in PubMed Commons below Cancer Res. 1990; 50: 3400-3407.

47. Na B, Huang Z, Wang Q, Qi Z, Tian Y, Lu CC. Transgenic expression of entire hepatitis B virus in mice induces hepatocarcinogenesis independent of chronic liver injury. See comment in PubMed Commons below PLoS One. 2011; 6: e26240.

48. Santantonio T, Jung MC, Schneider R, Fernholz D, Milella M, Monno L. Hepatitis B virus genomes that cannot synthesize pre-S2 proteins occur frequently and as dominant virus populations in chronic carriers in Italy. See comment in PubMed Commons below Virology. 1992; 188: 948-952.

49. Mimms L1. Hepatitis B virus escape mutants: “pushing the envelope” of chronic hepatitis B virus infection. See comment in PubMed Commons below Hepatology. 1995; 21: 884-887.

50. Liu S, Zhang H, Gu C, Yin J, He Y, Xie J. Associations between hepatitis B virus mutations and the risk of hepatocellular carcinoma: a meta-analysis. See comment in PubMed Commons below J Natl Cancer Inst. 2009; 101: 1066-1082.

51. Huy TT, Ushijima H, Win KM, Luengrojanakul P, Shrestha PK, Zhong ZH. High prevalence of hepatitis B virus pre-s mutant in countries where it is endemic and its relationship with genotype and chronicity. See comment in PubMed Commons below J Clin Microbiol. 2003; 41: 5449-5455.

52. Fernholz D, Galle PR, Stemler M, Brunetto M, Bonino F, Will H. Infectious hepatitis B virus variant defective in pre-S2 protein expression in a chronic carrier. See comment in PubMed Commons below Virology. 1993; 194: 137-148.

53. Le Seyec J, Chouteau P, Cannie I, Guguen-Guillouzo C, Gripon P. Role of the pre-S2 domain of the large envelope protein in hepatitis B virus assembly and infectivity. See comment in PubMed Commons below J Virol. 1998; 72: 5573-5578.

54. Wu CY, Chen YJ, Ho HJ, Hsu YC, Kuo KN, Wu MS. Association between nucleoside analogues and risk of hepatitis B virus–related hepatocellular carcinoma recurrence following liver resection. See comment in PubMed Commons below JAMA. 2012; 308: 1906-1914.

55. Zhang D, Dong P, Zhang K, Deng L, Bach C, Chen W. Whole genome HBV deletion profiles and the accumulation of preS deletion mutant during antiviral treatment. See comment in PubMed Commons below BMC Microbiol. 2012; 12: 307.

56. Pollicino T, Cacciola I2, Saffioti F2, Raimondo G3. Hepatitis B virus PreS/S gene variants: pathobiology and clinical implications. See comment in PubMed Commons below J Hepatol. 2014; 61: 408-417.

57. Kao JH, Liu CJ, Jow GM, Chen PJ, Chen DS, Chen BF. Fine mapping of hepatitis B virus pre-S deletion and its association with hepatocellular carcinoma. See comment in PubMed Commons below Liver Int. 2012; 32: 1373-1381.

58. Fang ZL, Sabin CA, Dong BQ, Wei SC, Chen QY, Fang KX. Hepatitis B virus pre-S deletion mutations are a risk factor for hepatocellular carcinoma: a matched nested case-control study. See comment in PubMed Commons below J Gen Virol. 2008; 89: 2882-2890.

59. Sinn DH, Choi MS, Gwak GY, Paik YH, Lee JH, Koh KC. Pre-s mutation is a significant risk factor for hepatocellular carcinoma development: a long-term retrospective cohort study. See comment in PubMed Commons below Dig Dis Sci. 2013; 58: 751-758.

60. Chen CH, Hung CH, Lee CM, Hu TH, Wang JH, Wang JC. Pre-S deletion and complex mutations of hepatitis B virus related to advanced liver disease in HBeAg-negative patients. See comment in PubMed Commons below Gastroenterology. 2007; 133: 1466-1474.

61. Su CW, Chiou YW, Tsai YH, Teng RD, Chau GY, Lei HJ. The Influence of Hepatitis B Viral Load and Pre-S Deletion Mutations on Post-Operative Recurrence of Hepatocellular Carcinoma and the Tertiary Preventive Effects by Anti-Viral Therapy. See comment in PubMed Commons below PLoS One. 2013; 8: e66457.

62. Huang HP, Hsu HY, Chen CL, Ni YH, Wang HY, Tsuei DJ. Pre-S2 deletions of hepatitis B virus and hepatocellular carcinoma in children. See comment in PubMed Commons below Pediatr Res. 2010; 67: 90-94.

63. Abe K, Thung SN, Wu HC, Tran TT, Le Hoang P, Truong KD. Pre-S2 deletion mutants of hepatitis B virus could have an important role in hepatocarcinogenesis in Asian children. See comment in PubMed Commons below Cancer Sci. 2009; 100: 2249-2254.

64. Tsai HW, Lin YJ, Lin PW, Wu HC, Hsu KH, Yen CJ, et al. A clustered ground-glass hepatocyte pattern represents a new prognostic marker for the recurrence of hepatocellular carcinoma after surgery. Cancer. 2011; 117: 2951-2960.

65. Shen FC, Su IJ, Wu HC, Hsieh YH, Yao WJ, Young KC. A pre-S gene chip to detect pre-S deletions in hepatitis B virus large surface antigen as a predictive marker for hepatoma risk in chronic hepatitis B virus carriers. See comment in PubMed Commons below J Biomed Sci. 2009; 16: 84.