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The Influence of Hepatitis B Viral Load and Pre-S Deletion Mutations on Mail-Operative Recurrence of Hepatocellular Carcinoma and the 3rd Preventive Effects by Anti-Viral Therapy

• Chien-Wei Su,
• Yu-Wei Chiou,
• Yi-Hsuan Tsai,
• Ruei-Dun Teng,
• Gar-Yang Chau,
• Hao-Jan Lei,
• Hung-Hsu Hung,
• Teh-Ia Huo,
• Jaw-Ching Wu

x

• Published: June 21, 2013
• https://doi.org/ten.1371/periodical.pone.0066457

Figures

Abstract

Background

Whether or not hepatitis B virus (HBV) genotypes, mutations, and viral loads decide outcomes for patients with HBV-induced hepatocellular carcinoma (HCC) remains controversial.

Aims

To study the influence of HBV viral factors on prognoses for patients with HBV-induced HCC after resection surgery and investigate if antiviral therapy could counteract the adverse effects of viral factors.

Methods

A full of 333 HBV-related HCC patients who underwent tumor resection were enrolled retrospectively. Serum HBV DNA levels, mutations, anti-viral therapy, and other clinical variables were analyzed for their association with mail service-operative recurrence.

Results

Afterwards a median follow-up of 45.9 months, 208 patients had HCC recurrence later resection. The 5-year overall survival and recurrence-free survival rates were 55.4% and 35.3%, respectively. Multivariate analysis showed indocyanine light-green retentiveness rate at 15 minutes >x%, gamma-glutamyltransferase (GGT) level >sixty U/L, macroscopic and microscopic venous invasion, and the absence of anti-viral therapy were significant hazard factors for recurrence. Anti-viral therapy could decrease recurrence in patients with early phase HCC, but the effect was less apparent in those with the Barcelona-Dispensary Liver Cancer stage C HCC. For patients without antiviral therapy afterward resection, serum HBV DNA levels >x^half-dozen copies/mL, GGT >60 U/Fifty, and macroscopic and microscopic venous invasion were meaning risk factors predicting recurrence. Amid the 216 patients without anti-viral therapy but with complete HBV surface gene mapping data, 73 were with pre-S deletion mutants. Among patients with college serum HBV Deoxyribonucleic acid levels, those with pre-S deletion had significantly higher rates of recurrence. Moreover, multivariate analysis showed multi-nodularity, macroscopic venous invasion, cirrhosis, advanced tumor prison cell differentiation, and pre-South deletion were significant adventure factors predictive of recurrence.

Conclusions

Ongoing HBV viral replication and pre-S deletion are crucial for determining postal service-operative tumor recurrence. Anti-viral therapy tin can assist reduce recurrence and ameliorate prognosis, especially for those with early phase HCC.

Commendation: Su C-W, Chiou Y-W, Tsai Y-H, Teng R-D, Chau K-Y, Lei H-J, et al. (2013) The Influence of Hepatitis B Viral Load and Pre-S Deletion Mutations on Post-Operative Recurrence of Hepatocellular Carcinoma and the Tertiary Preventive Furnishings by Anti-Viral Therapy. PLoS One viii(six): e66457. https://doi.org/x.1371/periodical.pone.0066457

Editor: John Luk, Johnson & Johnson Medical, China

Received: March 12, 2013; Accustomed: May 6, 2013; Published: June 21, 2013

Copyright: © 2013 Su et al. This is an open-access commodity distributed under the terms of the Creative Commons Attribution License, which permits unrestricted employ, distribution, and reproduction in any medium, provided the original writer and source are credited.

Funding: This work was supported by grants from the National Science Quango of Taiwan (NSC 101-2314-B-075-013-MY2), Taipei Veterans General Hospital (V95C1-014, VGHUST100-G7-2-1), Yang-Ming Academy (101AC-T501, Ministry building of Education, Aim for the Top University Plan), and the Center of Excellence for Cancer Inquiry at TVGH (DOH101-TD-C-111-007), Taipei, Taiwan. The funders had no role in study design, information collection and analysis, decision to publish, or training of the manuscript.

Competing interests: The authors accept declared that no competing interests exist.

Introduction

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide [1]. Surgical resection is the major treatment modality for patients with early-stage HCC and well-preserved liver function [1]–[3]. Even so, the long-term issue after resection remains unsatisfactory considering of the high post-operative recurrence rate, which accounts for the high bloodshed [4], [5]. To improve prognosis, risk factors affecting hepatocarcinogenesis and mail service-surgical recurrence of HCC must exist identified.

Tumor size and number, presence of venous invasion, and liver functional reserve are reported to determine post-operative tumor recurrence [two], [half-dozen], [7]. Equally regards viral factors, hepatitis B virus (HBV) is the well-nigh mutual etiological factor of HCC, especially in Asia [one], [viii]. The pathogenesis of HCC in chronic HBV infections has been investigated comprehensively in recent years. The REVEAL-HBV cohort study demonstrates a significant dose-response relationship between serum HBV Deoxyribonucleic acid levels and the chance of developing HCC in chronic HBV carriers [nine]. In addition to viral load, HBV genotype and basal cadre promoter (BCP) mutations are also correlated with the development of HCC [10], [xi]. However, whether or not HBV genotypes, mutations, and viral loads determine outcomes for patients with HBV-induced HCC after resection surgery remains controversial [2], [12]. Moreover, the efficacy of anti-viral therapy afterwards resection is still debated [13], [14].

There are iii forms of hepatitis B surface antigen (HBsAg) limerick: big (coded for by the pre-S1/pre-S2/Due south gene), middle (the preS2/S gene), and modest (the S gene) protein [xv]. The pre-Due south region of the HBsAg carries several potent T and B cell epitopes [16]. During the natural history of chronic hepatitis B, viral variants and deletion mutants may exist selected by immune response. Cross-sectional studies take demonstrated that patients with HBV-induced HCC had significantly higher frequencies of harboring pre-S deletion mutants than HBV carriers without HCC [17], [18]. Longitudinal cohort studies likewise testify that the presence of pre-S deletion mutants in chronic hepatitis B patients is a significant risk factor of developing HCC during follow-upwards [19]. While these reports propose a close human relationship between pre-S deletion mutants and hepatocarcinogenesis, the prognostic value of pre-S deletion mutants in HCC patients afterwards curative therapies is still unclear due to the relatively small size of patient number with pre-S deletion mutants in the previous studies [xx], [21].

This study aimed to comprehensively elucidate the touch of ongoing HBV viral replication and viral mutations on postoperative HCC recurrence, and the third chemo-preventive effects of anti-viral therapy on recurrence.

Materials and Methods

Patients and Follow-upward

There were 607 consecutive treatment-naïve HBV-related HCC patients who underwent curative resection surgery in Taipei Veterans General Hospital from 1990 to 2007. The inclusion criteria were (a) positive hepatitis B surface antigen (HBsAg) in sera; (b) liver function of A or B by Kid'south classification, with an indocyanine green 15-minute retention charge per unit (ICG-15R) <xxx%; (c) tumors involving no more than three Healey's segment without portal vein master trunk interest; (d) absence of other major diseases that might complicate surgery; (e) absenteeism of extra-hepatic tumor dissemination [half-dozen], [22]. Amongst them, 333 patients who had stored serum samples available for virological assay were enrolled in this study. The demographic characteristics betwixt patients with and those without virological data were compared in Tabular array S1. The demographic characteristics were similar betwixt these two groups except patients who did not have item virological data had larger tumor size than those with detail HBV virological data. No other forms of adjuvant anti-tumor therapy such as local ablation therapy, chemoembolization, or molecular target therapy, were performed before or afterwards resection until the emergence of tumor recurrence. Patients with concurrent infection of hepatitis C virus (HCV) or hepatitis D virus (HDV) were excluded.

Afterwards surgery, the macroscopic features of the tumor, including size, number, and vascular invasion were assessed. Histologically, microscopic vascular invasion and cirrhosis of the non-tumor part were examined by microscopy. Cancer cell differentiation was determined by Edmondson grade [23].

Afterward surgery, patients visited outpatient clinics regularly every 3 months and assessed by testing serum liver biochemistries and AFP levels, and ultrasonography. Tumor recurrence was suspected if serum alpha-fetoprotein (AFP) levels were elevated (>20 ng/mL) or if new lesions were detected by surveillance ultrasonography. These lesions were further diagnosed by dynamic computed tomography or magnetic resonance imaging with presence of typical vascular pattern for HCC.

The Institutional Review Board of the Taipei Veterans General Infirmary has approved the study protocol and consent procedure, which complied with the standards of the Declaration of Helsinki. All of the patients provided their written informed consent to participate in this study.

Biochemical and Serological Markers

Serum HBsAg levels were quantified past Architect HBsAg QT (Abbott Diagnostic, Germany) according to the manufacturer instructions. Hepatitis B e antigen (HBeAg), antibiotic against HBeAg (anti-HBe), and antibody to HDV were tested using a radio-immunoassay kit (Abbott Laboratories, N Chicago, IL), while anti-HCV was assessed by a second-generation enzyme immunoassay kit (Abbott Laboratories, Due north Chicago, IL).

Serum biochemistries were measured using the Roche/Hitachi Modular Analytics Systems (Roche Diagnostics GmbH, Mannheim, Germany) and serum AFP level was tested by radio-immunoassay (Serono Diagnostic SA, Coinsin/VD, Switzerland).

Detection and Quantification of HBV Deoxyribonucleic acid, HBV Genotyping and Sequencing

The complete HBV envelope open-reading frame was amplified using a pair of primers B2822(+) (5′-ggg,TCA,CCA,TAT,TCT,Tgg-3′) and B840R (5′- ACC,CCA,TCT,TTT,TgT,TTT,gTT,Agg-3′) that covered the entire coding region of pre-S1, pre-S2, and S gene (2848–3215 and 1–835) via polymerase concatenation reaction (PCR). The PCR was performed in a thermal cycler (Perkin Elmer Cetus Corp., Norwalk, CT), beginning at 95°C for 5 min, followed past 35 cycles (each cycle: 95°C for 40 sec, 55°C for 40 sec, 72°C for 2 min) of amplification, and ending at 72°C for 10 min. The PCR products were analyzed in ane.5% agarose gel, followed by ethidium bromide staining.

The amplified PCR products were ligated into the plasmid pCR2 vector (Original TA cloning Kit, Invitrogen Corporation, Carlsbad, CA) according to the manufacturer's didactics. The ligation mixture was used to transform the competent Escherichia coli strain DH5α (Gibco BRL, Life Technologies, Gaithersburg, MD) and incubated overnight in LB agar plate containing 100 µg/ml ampicillin and 30 µg/ml X-gal at 37°C. The successful ligation clones in bluish white screening were picked-up and cultured overnight in three ml LB-Amp goop at 37°C. Plasmid Deoxyribonucleic acid was purified past QIAprep Spin MiniPrep Kit (QIAGEN GmbH, D-40724 Hilden). Sequencing of pre-S1, pre-S2, and South gene was performed with dye terminator bike sequencing kit (Dye terminator cycle sequencing core kit #402117, Perkin Elmer Cetus Corp., Norwalk, CT) according to the manufacturer's instruction and sequencing products were analyzed in an ABI 373A sequencer (Perkin Elmer Cetus Corp., Norwalk, CT).

The HBV DNA levels were measured by a Cobas Amplicor HBV monitor (Roche Diagnostic Organization, Basel, Switzerland), with a detection limit of 300 copies/mL. In this test, five.82 copies/mL was equal to 1 IU/mL. Genotyping of HBV was performed by PCR restriction fragment length polymorphism of the surface gene of HBV and was further verified by sequencing as previously described [two].

Statistical Analysis

All statistical analyses were performed using the SPSS 17.0 for Windows (SPSS. Inc., Chicago, IL, USA). _ENREF_18Pearson chi-square analysis or Fisher's exact test were used to compare categorical variables, while continuous variables were compared using the Isle of mann-Whitney U-test. Cumulative recurrence rate or overall survival rates were estimated past the Kaplan-Meier method and compared using the log-rank exam. Variables with statistical significance (p<0.05) or proximate to it (p<0.1) past univariate analysis underwent multivariate analysis by forward stepwise Cox regression model. A two-tailed p<0.05 was considered statistically significant.

Results

Baseline Clinical Demographics, Biochemical, Surgical, Virological, and Pathological Data of all Patients

The 333 cases had a median age of 56 years and median tumor size of 4.0 cm. The criteria for the indication of reimbursed antiviral therapy for chronic hepatitis B in Taiwan were every bit the followings: (1) for cirrhotic patients, serum HBV Dna levels >2000 IU/mL irrespective of serum alanine aminotransferase (ALT) levels; (2) for non-cirrhotic patients, serum ALT levels >fourscore U/L in improver to serum HBV Deoxyribonucleic acid levels >20000 IU/mL in HBeAg-positive patients and HBVDNA levels >2000 IU/mL in HBeAg-negative patients, respectively. As reimbursed anti-viral therapy was implemented in Taiwan since 2003; therefore, only 62 (18.6%) patients received anti-viral therapy later on resection, including 40 with lamivudine, nineteen with entecavir, and 3 with pegylated interferon. The demographic characteristics between patients with and those without anti-viral therapy after resection surgery are shown in Table S2.

The HBV genotype distribution was one genotype A, 174 genotype B, 139 genotype C, and 19 unclassified. Among the unclassified HBV genotypes, 9 were due to undetectable viral genomes, while the remaining ten were due to weak signals. Compared to genotype B, patients with genotype C HBV had lower serum albumin levels, lower platelet counts, college serum ALT and aspartate aminotransferase (AST) levels, higher ICG-15R, and prothrombin fourth dimension international normalized ratio values ( Table 1 ). Moreover, prevalence of cirrhosis was higher in patients with genotype C HBV.

Regarding viral factors, patients with genotype C HBV had a college prevalence of positive HBeAg in sera and BCP mutation, and lower frequency of pre-cadre mutation (G1896A) than those with genotype B, merely serum HBV Deoxyribonucleic acid and HBsAg levels were comparable between these two groups. Tumor characteristics were not significantly different between the ii groups.

Factors Associated with Poor Overall Survival and Recurrence afterward Resection

Subsequently a median follow-upward of 45.9 months (25^th-to-75^th percentile range, 22.4–78.9 months), 165 patients died. The overall cumulative survival rates at 1, 3, and 5 years were 86.7%, 69.3%, and 55.4%, respectively. Moreover, 208 patients had tumor recurrence. The cumulative recurrence-free rates at i, 3, and 5 years were 66.vii%, 44.7%, and 35.3%, respectively.

Univariate analysis of risk factors associated with overall survival and recurrence were shown in Tabular array S3 and Tabular array S4, respectively. Past multivariate assay, ICG-15R >10%, alkaline phosphatase (Alk-P) >100 U/L, macroscopic venous invasion, microscopic venous invasion, and the absence of anti-viral therapy were significant risk factors predictive of poor overall survival (Table S5). Furthermore, ICG-15R >10%, gamma-glutamyltransferase (GGT) >lx U/L, macroscopic venous invasion, microscopic venous invasion, and the absenteeism of anti-viral therapy were significant adventure factors for tumor recurrence after resection ( Table ii ). Consequently, anti-viral therapy was a critical factor for determining post-operative prognosis both in terms of overall survival and recurrence ( Figure 1 ).

Figure 1. The impact of anti-viral therapy and tumor stage on post-operative prognosis.

Patients who received anti-viral therapy after resection had (A) higher overall survival charge per unit (p<0.001) and (B) lower recurrence charge per unit (p<0.001) than those who did not receive anti-viral therapy. Moreover, patients in BCLC phase C HCC had lower overall survival rate (C) and higher recurrence charge per unit (D) than those in BCLC stage A or B.

https://doi.org/10.1371/journal.pone.0066457.g001

The effect of Anti-viral Therapy Stratified by the Barcelona-Clinic Liver Cancer (BCLC) Stage and viral Factors

Equally the demographic characteristics were non perfectly match betwixt patients with and those without antiviral therapy later resection. Beside multivariate analysis, we further assessed the efficacy of anti-viral therapy stratified by tumor stage and viral factors to diminish the impact of confounding factors on prognosis. Patients with BCLC tumor stage C HCC had significantly lower overall survival rate and higher recurrence rate than those with phase A or B ( Figure 1 ). Moreover, anti-viral therapy could meliorate the post-operative prognosis both in terms of overall survival and recurrence in patients with early stage HCC, but the outcome was less apparent in those with BCLC stage C ( Figure 2 ).

Figure 2. The event of anti-viral therapy on mail service-operative prognosis stratified by tumor stage.

Among patients with early phase HCC, those who received anti-viral therapy after resection had higher overall survival charge per unit (A, BCLC stage A, p = 0.001; B, BCLC stage B, p<0.001) and lower recurrence charge per unit (C, BCLC stage A, p = 0.045; D, BCLC stage B, p = 0.009) than their counterpart. For patients with BCLC stage C HCC, the overall survival rate were comparable between those with and without antiviral therapy (E, p = 0.158); but patients receiving anti-viral therapy later resection had a trend of lower recurrence rate than those who did not receive anti-viral therapy (F, p = 0.065).

https://doi.org/x.1371/periodical.pone.0066457.g002

Regarding viral factors, anti-viral therapy could reduce post-operative recurrence both in the setting of high and depression serum HBV Deoxyribonucleic acid levels ( Figure 3A–B ). Similarly, the recurrence rates were both lower in patients receiving anti-viral therapy than their analogue irrespective of their serum HBsAg levels ( Figure 3C–D ).

Figure 3. The impact of anti-viral therapy on post-operative recurrence stratified by viral factors.

Patients who received anti-viral therapy later on resection had significantly lower recurrence charge per unit both in the setting of serum HBV DNA levels >ten⁵ copies/mL (A, p<0.001) and ≤10^five copies/mL (B, p = 0.038). (C) Among patients with serum HBsAg >500 IU/mL, anti-viral therapy was associated with lower recurrence rate (p = 0.001). (D) In patients with serum HBsAg ≤500 IU/mL, the recurrence rates were too lower in patients receiving antiviral therapy after resection surgery (p = 0.037).

https://doi.org/x.1371/journal.pone.0066457.g003

Factors Associated with Overall Survival and Recurrence in HCC Patients without Anti-viral Therapy

As anti-viral therapy might confound the impact of viral factors, to eliminate this bias, 62 patients who received anti-viral therapy later resection surgery were excluded and the remaining 271 patients were further analyzed for prognostic factors.

By multivariate analysis, serum HBVDNA levels >10^vi copies/mL, GGT >60 U/50, macroscopic venous invasion, and microscopic venous invasion were significant take chances factors predictive of tumor recurrence after resection ( Table two ). The result of multivariate assay of significant take chances factors associated with poor overall survival is shown in Table S5.

Factors Associated with Poor Prognosis in the HCC Sub-group with Available Complete HBs Gene Sequence Data just without Anti-viral Therapy

Among the 216 patients with sufficient serum samples for consummate HBs cistron sequencing, 73 had the pre-S deletion HBV mutants, including fifteen with the pre-S1 deletion mutant only, 43 with the pre-S2 deletion mutant just, and xv with both.

Of the 58 patients with the pre-S2 deletion mutants HBV, 25 (43.1%) had the deletion that ended at amino acid 142 of the pre-S region. Proline was the preserved amino acid on this site in all of these mutants.

The demographic characteristics, including tumor factors, liver functional reserve and virological factors (genotype, serum HBVDNA and HBsAg levels, proportion of BCP and pre-core mutations) were like between patients with pre-S1 deletion mutants and those with pre-S2 deletion mutants.

Comparison the demographic characteristics between patients with and those without the pre-Southward deletion HBV mutants, those with the pre-Due south deletion mutants had a higher charge per unit of liver cirrhosis on the non-tumor function, lower serum albumin levels, higher GGT levels, and a trend of college ALT level ( Table 3 ). These imply that HCC patients with the pre-South deletion mutants of HBV had more advanced hepatic fibrosis and poorer liver functional reserve. Regarding viral factors, the proportion of positive HBeAg in sera and genotype C were higher in patients infected with the pre-S deletion HBV mutants than those without the pre-Southward deletion mutants. Moreover, patients with the pre-S deletion had a tendency of higher serum HBVDNA levels (p = 0.055). Tumor factors were comparable between the two groups.

Univariate analysis of hazard factors that correlated with overall survival and recurrence were shown in Table S6 and Table S7, respectively. By multivariate analysis, multi-nodularity, presence of macroscopic venous invasion, cirrhosis, advanced tumor cell differentiation, and pre-S deletion were the significant risk factors predictive of post-resection recurrence ( Tabular array ii ). As well, the recurrence-free survival rates were comparable betwixt patients with pre-S1 deletion mutants and those with pre-S2 deletion mutants.

Impact of Pre-Southward deletion on Post-operative Recurrence Stratified by Serum HBV DNA and HBsAg Levels

The effects of pre-S deletion in mail-operative recurrence as stratified by serum HBV viral load and HBsAg levels were assessed. For patients with higher serum HBV Dna (>10^five copies/mL) or higher HBsAg level (>500 IU/mL), the presence of pre-South deletion was significantly associated with a college rate of recurrence ( Figure 4 ). However, for those with low viral loads or lower HBsAg levels, recurrence rates were comparable between patients with and those without pre-South deletion.

Figure 4. Comparing of post-resection recurrence between patients with and those without pre-S deletion mutants stratified by serum HBVDNA and HBsAg levels.

(A) Among patients with serum HBVDNA >ten⁵ copies/mL, the presence of pre-S deletion correlated with a college charge per unit of mail service-resection recurrence than patients without the pre-S deletion (p = 0.028). (B) Among patients with serum HBsAg >500 IU/mL, pre-S deletion was associated with college recurrence rate later on resection (p = 0.047). (C) In patients with serum HBVDNA ≤10^v copies/mL, the recurrence rates were comparable between patients with and those without pre-S deletion (p = 0.926). (D) In patients with serum HBsAg ≤500 IU/mL, the recurrence rates were similar betwixt patients with and those without pre-S deletion (p = 0.253).

https://doi.org/ten.1371/journal.pone.0066457.g004

Give-and-take

For patients with HBV-induced HCC, ongoing HBV viral replication may initiate tumor recurrence after resection through several mechanisms [ii], [fourteen]. Firstly, it can induce persistent hepatic inflammation, oxidative stress, and fibrosis, which in turn leads to hepatocarcinogenesis. However, it may need longer time to accumulate sufficient mutations for tumor recurrence simply by chronic inflammation. Our previous study shows that the grade of hepatic inflammation is crucial for the development of tardily recurrence (recurrence occurring after two years of resection surgery) [2]. 2d, information technology may also have direct mutagenic effects through the integration of the HBV Dna into the host genome, initiation of oncogenes mediated by prolonged expression of viral proteins, and inhibition of tumor suppressor genes like p53 [24], [25]. One contempo genome-broad survey for HBV-induced HCC demonstrates that HBV integration occurs more oftentimes in tumors (86.iv%) than in the side by side liver tissues (30.seven%). [25] Moreover, the HBV integration breakpoints seem to exist associated with increased copy-number variations and chromosome instability.

Because anti-viral therapy can suppress HBV replication, meliorate liver inflammation and fibrosis, and meliorate liver functional reserve, meta-analysis of previous studies reveals that anti-viral therapy improves the overall survival of HCC patients later on curative therapy [26]. However, its role in reducing recurrence rate is still controversial. This may be due to differences in demographic characteristics, liver functional reserve, and tumor factors in previous reports. The patient numbers are likewise relatively smaller in these studies [26], [27]. A recent report using National Wellness Insurance Research Dataset suggested that antiviral therapy might reduce postoperative HCC recurrence [28]. However, the dataset did not provide detail analysis of HBV loads, HBsAg levels and HBV mutations that are closely linked to the evolution of HCC [ix], [10], [29], [thirty].

In this cohort, for those without anti-viral therapy later on resection, college serum HBV DNA level was an important risk cistron associated with recurrence. Of note, anti-viral therapy tin can amend the overall survival rate and reduce recurrence afterwards resection even in patients with high viral loads and HBsAg levels and the latter two factors were not significantly associated with recurrence in the presence of antiviral therapy. Antiviral therapy should be recommended as tertiary preventive measure for the reduction of postoperative HCC recurrence in patients with HBV-related HCC.However, the impact of anti-viral therapy on improving post-operative outcomes was less significant in patients with BCLC stage C. Information technology may be attributed to that tumor factors boss the prognoses in patients with advanced stage HCC fifty-fifty subsequently curative therapies, thereby diminish the effect of anti-viral therapy in improving the "field factors" of not-tumor part [2], [27].

The pre-Due south region of HBV is crucial in mediating the attachment of the virus to host hepatocytes. Moreover, it is important for interacting with the host immune responses [17]. It has been reported that the proportions of pre-Southward deletion mutants are significantly higher in patients with HBV-related HCC than in those with inactive HBV carrier status, implying that these mutants may exist associated with HCC carcinogenesis [17], [nineteen]. Previous studies show that the deletion mutants in the pre-Due south region tin cause the accumulation of large surface proteins in the endoplasmic reticulum (ER) and induce ER stress, which in turn provokes mutagenesis through the generation of oxidative stress, cyclooxygenase-2 expression, and DNA damages [17], [31]. 1 recent study further demonstrates that the ER stress induced by pre-S mutants leads to disturbances in cyclin A expression and centrosome over-duplication, thereby causing chromosome instability and hepatocarcinogenesis [31]. Even so, there have been few reports about the influence of pre-S deletion mutants on postoperative HCC recurrence. Our electric current study indicates that for patients with HBV-related HCC, the presence of pre-S deletion mutants is an important factor for predicting mail-operative HCC recurrence. This implies the critical role of pre-S deletion mutants of HBV both in hepatocarcinogenesis and tumor recurrence.

In this study, patients with pre-Due south deletion mutants have a higher proportion of positive HBeAg in sera and a tendency of higher HBV Deoxyribonucleic acid levels than their counterpart in the present cohort. As most of the deletion regions encompassed T and B cell epitopes, information technology is likely that pre-S deletion HBV mutants may escape more hands from host immune clearance, resulting in higher viral replication than those without pre-Due south deletion [19]. Of note, the large number of patients in this study which provided subgroup assay firstly reported that the presence of pre-S deletion mutants correlates to a higher charge per unit of tumor recurrence only in patients with serum HBV DNA levels >10^five copies/mL or HBsAg >500 IU/mL. In contrast, the recurrence rates are like between patients with and those without pre-S deletion in patients with low viral loads. Moreover, for patients receiving antiviral therapy afterwards resection, the prognoses were comparable betwixt patients with and those without pre-S deletion mutants (Figure S1). This implies that the ER and oxidative stresses caused by the accumulation of large surface protein may be more credible in the setting of high HBV DNA levels. In other words, the hepatocarcinogenesis and tumor recurrence induced past pre-S deletion mutants may be based on active HBV viral replication, the accumulation of oxidative stress, and subsequent chromosomal instability [31].

In conclusion, on-going viral replication and the presence of pre-Due south deletion are both crucial predictors of mail-operative HCC recurrence. Anti-viral therapy can reduce post-operative recurrence and improve prognosis, particularly for those with early phase HCC.

Supporting Information

Figure S1.

The touch of pre-S deletion mutants on mail service-operative recurrence for patients receiving anti-viral therapy. The recurrence rate after resection surgery were comparable betwixt patients with and those without pre-S deletion mutants (p = 0.161).

https://doi.org/ten.1371/journal.pone.0066457.s001

(TIF)

Acknowledgments

The authors thank Mrs. Pui-Ching Lee for her assistance with the statistical analysis.

Author Contributions

Conceived and designed the experiments: CWS GYC HHH JCW. Performed the experiments: CWS YWC YHT RDT JCW. Analyzed the information: CWS HJL HHH TIH JCW. Contributed reagents/materials/analysis tools: CWS YWC YHT RDT JCW. Wrote the paper: CWS JCW.

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Source: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0066457

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