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Background: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality worldwide. It is a highly heterogeneous disease with poor prognosis and limited treatment options, which highlights the need for reliable biomarkers. This study aims to explore molecular markers that allow stratification of HCC and may lead to better prognosis and treatment prediction., Materials and Methods: We studied 20 candidate genes (HCC hub genes, potential drug target genes, predominant somatic mutant genes) retrieved from literature and public databases with potential to be used as the molecular markers. We analysed expression of the genes by RT-qPCR in 30 HCC tumour and adjacent non-tumour paired samples from Vietnamese patients. Fold changes in expression were then determined using the 2 -∆∆CT method, and unsupervised hierarchical clustering was generated using Cluster v3.0 software., Results: Clustering of expression data revealed two subtypes of tumours (proliferative and normal-like) and four clusters for genes. The expression profiles of the genes TOP2A, CDK1, BIRC5, GPC3, IGF2, and AFP were strongly correlated. Proliferative tumours were characterized by high expression of the c-MET, ARID1A, CTNNB1, RAF1, LGR5, and GLUL1 genes. TOP2A, CDK1, and BIRC5 HCC hub genes were highly expressed (> twofold) in 90% (27/30), 83% (25/30), and 83% (24/30) in the tissue samples, respectively. Among the drug target genes, high expression was observed in the GPC3, IGF2 and c-MET genes in 77% (23/30), 63% (19/30), and 37% (11/30), respectively. The somatic mutant Wnt/ß-catenin genes (CTNNB1, GLUL and LGR5) and TERT were highly expressed in 40% and 33% of HCCs, respectively. Among the HCC marker genes, a higher percentage of tumours showed GPC3 expression compared to AFP expression [73% (23/30) vs. 43% (13/30)]., Conclusion: The custom panel and molecular markers from this study may be useful for diagnosis, prognosis, biomarker-guided clinical trial design, and prediction of treatment outcomes., (© 2022. The Author(s).)

Background: Programmed cell death-1 (PD-1) variants and circulating level of soluble PD-1 are associated with susceptibility to malignant and infectious disease. This study aimed to examine the association of PD-1.5 and PD-1.9 variants, and plasma sPD-1 level with hepatitis B virus (HBV) infection and disease progression., Methods: The study cohort consisted of adults infected with HBV (n=513) - stratified by clinical course, including chronic hepatitis B (CHB, n=173), liver cirrhosis (LC, n=134) and hepatocellular carcinoma (HCC, n=206) - and matched healthy controls (HC, n=196). The PD-1.5 (rs2227981 C/T) and PD-1.9 (rs2227982 C/T) genetic variants were genotyped by Sanger sequencing, and plasma sPD-1 levels were quantified by enzyme immunoassay., Results: Plasma sPD-1 levels were significantly higher among patients infected with HBV. The highest plasma sPD-1 levels were observed in patients with CHB, followed by patients with LC and HCC. In addition, the plasma sPD-1 levels correlated positively with liver inflammation [aspartate transaminase (AST): rho=0.57, P<0.0001; alanine aminotransferase: rho=0.57, P<0.0001], and were positively correlated with liver fibrosis [AST to platelet ratio index score: rho=0.53, P<0.0001). The PD-1.9 TT genotype was less common in patients with CHB compared with patients with LC, HCC, and HCC+LC in both codominant and recessive models (P<0.01), and was found to be a risk factor for HCC predisposition {HCC vs non-HCC: odds ratio (OR) 2.0 [95% confidence interval (CI) 1.13-3.7], P adj =0.017}. The PD-1.5 CT genotype was associated with reduced risk of acquiring HCC [OR 0.6 (95% CI 0.4-0.9), P adj =0.031]., Conclusion: sPD-1 level was associated with liver inflammation and progression of liver fibrosis, and the PD-1.5 and PD-1.9 variants were associated with HBV infection and progression of liver disease., Competing Interests: Conflict of interest statement None declared., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The inhibitory effects of programmed cell death 1/programmed cell death ligand 1 (PD-1/PD-L1) modulates T-cell depletion. T-cell depletion is one of the key mechanisms of hepatitis B virus (HBV) persistence, in particular liver disease progression and the development of hepatocellular carcinoma (HCC). This case-control study aimed to understand the significance of PD-1 polymorphisms (PD-1.5 and PD-1.9) association with HBV infection risk and HBV-induced liver disease progression. Genotyping of PD-1.5 and PD-1.9 variants was performed by direct Sanger sequencing in 682 HBV-infected patients including chronic hepatitis (CHB, n = 193), liver cirrhosis (LC, n = 183), hepatocellular carcinoma (HCC, n = 306) and 283 healthy controls (HC). To analyze the association of PD-1 variants with liver disease progression, a binary logistic regression, adjusted for age and gender, was performed using different genetic models. The PD-1.9 T allele and PD-1.9 TT genotype are significantly associated with increased risk of LC, HCC, and LC + HCC. The frequencies of PD-1.5 TT genotype and PD-1.5 T allele are significantly higher in HCC compared to LC patients. The haplotype CT (PD-1.5 C and PD-1.9 T) was significantly associated with increased risk of LC, HCC, and LC + HCC. In addition, the TC (PD-1.5 T and PD-1.9 C) haplotype was associated with the risk of HCC compared to non-HCC. The PD-1.5 CC, PD-1.9 TT, genotype, and the CC (PD-1.5 C and PD-1.9) haplotype are associated with unfavorable laboratory parameters in chronic hepatitis B patients. PD-1.5 and PD1.9 are useful prognostic predictors for HBV infection risk and liver disease progression.

Telomerase reverse-transcriptase (TERT) gene promoter mutations in circulating cell-free DNA (cfDNA) as well as the levels of circulating microRNA-122 (miR-122) have been reported as potential noninvasive biomarkers for several. This study evaluates the diagnostic performance of potent biomarker-based panels composing of serological AFP, miR-122 and circulating TERT promoter mutations for screening HBV-related HCC. TERT promoter mutations (C228T and C250T) and miR-122 expression were assessed in the plasma samples from 249 patients with HBV-related liver diseases by nested PCR and qRT-PCR assays, respectively. The diagnostic values of TERT promoter mutations, miR-122 expression and biomarker-based panels were assessed by computation of the area under the curve (AUC). Nested-PCR assays were optimized to detect C228T and C250T mutations in TERT promoter with detection limit of 1%. The common hotspot C228T was observed in 22 HCC cases. The triple combinatory panel (AFP@TERT@miR-122) acquired the best diagnostic value to distinguish HCC from CHB (AUC = 0.98), LC (AUC = 0.88) or non-HCC (LC + CHB, AUC = 0.94) compared to the performance of double combinations or single biomarkers, respectively. Notably, among patients with AFP levels≤20 ng/μl, the double combination panel (TERT@miR-122) retains satisfactory diagnostic performance in discriminating HCC from the others (HCC vs. CHB, AUC = 0.96; HCC vs. LC, AUC = 0.88, HCC vs. non-HCC, AUC = 0.94). The triple combination panel AFP@TERT@miR-122 shows a better diagnostic performance for screening HCC in HBV patients, regardless of AFP levels. The newly established panels can be a potential application in clinical practice in Vietnamese setting.

SMYD3 (SET and MYND domain-containing protein 3) is involved in histone modification, which initiates oncogenesis by activating transcription of multiple downstream genes. To investigate associations of variable numbers of tandem repeats (VNTR) variants in the SMYD3 gene promoter, SMYD3 serum levels and SMYD3 mRNA expression in hepatitis B virus (HBV) infection and clinical progression of related liver disease. SMYD3 VNTRs were genotyped in 756 HBV patients and 297 healthy controls. SMYD3 serum levels were measured in 293 patients and SMYD3 mRNA expression was quantified in 48 pairs of hepatocellular tumor and adjacent non-tumor liver tissues. Genotype SYMD3 VNTR 3/3 was more frequent among HCC patients than in controls (P adjusted  = 0.037). SMYD3 serum levels increased according to clinical progression of liver diseases (P = 0.01); HCC patients had higher levels than non-HCC patients (P = 0.04). Among patients with SMYD3 VNTR 3/3, HCC patients had higher SMYD3 levels than others (P < 0.05). SMYD3 mRNA expression was up-regulated in HCC tumor tissues compared to other tissues (P = 0.008). In conclusion, upregulation of SMYD3 correlates with the occurrence of HCC and SMYD3 VNTR 3/3 appears to increase the risk of HCC through increasing SMYD3 levels. SMYD3 may be an indicator for HCC development in HBV patients.

Background : ISGylation is the conjugation of ISG15 with target proteins. ISGylation occurs through an enzymatic cascade, which is similar to that of ubiquitination. Through ISGylation, ISG15 can bind to proteins involved in cell proliferation and differentiation, thus promoting genesis and progression of malignancies. The present study aims to investigate expression of genes involved in ISGylation and ubiquitination in patients with hepatocellular carcinoma and to correlate gene expression with clinical laboratory parameters of these patients. Methods : mRNA expression of genes encoding enzymes involved in the ISGylation process ( EFP , HERC5, UBA1, UBC and USP18 ) was evaluated by quantitative real-time PCR in 38 pairs of tumour and adjacent non-tumour tissues from patients with hepatocellular carcinoma and correlated with distinct clinical laboratory parameters. Results: Relative mRNA expression of EFP , HERC5, UBA1 and USP18 was significantly higher in tumour tissues compared to adjacent non-tumour tissues ( P =0.006; 0.012; 0.02 and 0.039, respectively). The correlation pattern of mRNA expression between genes in the tumours differed from the pattern in adjacent non-tumour tissues. Relative expression of EFP , HERC5 and UBA1 in adjacent non-tumour tissues was positively associated with direct bilirubin levels (Spearman's rho=0.31, 0.33 and 0.45; P =0.06, 0.05 and 0.01, respectively) and relative expression of USP18 in adjacent non-tumour tissues correlated negatively with ALT levels (Spearman's rho= -0.33, P =0.03). Conclusions : EFP , HERC5, UBA1, and USP18 genes are upregulated in tumour tissues of patients with HCC and, thus, may be associated with the pathogenesis of hepatocellular carcinoma., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Vitamin D derivatives and their receptor (VDR) are potent modulators of immune responses in various diseases including malignancies as well as in metabolic and infectious disorders. The impact of vitamin D receptor polymorphisms on clinical outcomes of hepatitis B virus (HBV) infection is not well understood. This study aims to investigate the potential role of VDR polymorphisms (TaqI, FokI, ApaI, and BsmI) in Vietnamese HBV infected patients and to correlate these polymorphisms with the progression of HBV-related liver disease., Methods: Four hundred forty-three HBV infected patients of the three clinically well-defined subgroups chronic hepatitis B (CHB, n = 183), liver cirrhosis (LC, n = 89) and hepatocellular carcinoma (HCC, n = 171) and 238 healthy individuals (HC) were enrolled. VDR polymorphisms were genotyped by DNA sequencing and in-house validated ARMS assays. Logistic regression models were applied in order to determine the association of VDR polymorphisms with manifest HBV infection as well as with progression of related liver diseases mulin different genetic models., Results: The VDR ApaI CA genotype was less frequent in HCC than in CHB patients in different genetic models (codominant model, OR = 0.5, 95%CI = 0.3-0.84, P = 0.004; dominant model, OR = 0.46, 95%CI = 0.27-0.76, P = 0.0023). In the recessive model, the genotype ApaI AA was found more frequently among HCC compared to CHB patients (OR = 2.56, 95%CI = 1.01-6.48, P = 0.04). Similarly, the ApaI CA genotype was less frequent in HCC than in non-HCC group codominant model, OR = 0.6, 95%CI = 0.4-0.98, dominant model, P = 0.04 and OR = 0.6, 95%CI = 0.38-0.90, P = 0.017). The ApaI genotypes CA and AA was significantly associated with higher levels of liver enzymes, bilirubin, and HBV DNA (P < 0.05). No association between TaqI, FokI and BsmI polymorphisms and any clinical outcome as well as liver disease progression was found., Conclusions: Among the four investigated VDR polymorphisms, ApaI is associated with clinical outcome and liver disease progression in Vietnamese HBV infected patients.

Background: Clinical progression of HBV-related liver diseases is largely associated with the activity of HBV-specific T cells. Soluble fibrinogen-like protein 2 (sFGL2), mainly secreted by T cells, is an important effector molecule of the immune system., Methods: sFGL2 levels were determined by ELISA assays in sera of 296 HBV patients clinically classified into the subgroups of acute hepatitis B (AHB), chronic hepatitis B (CHB), liver cirrhosis (LC), hepatocellular carcinoma (HCC) and patients with LC plus HCC. As control group, 158 healthy individuals were included. FGL2 mRNA was quantified by qRT-PCR in 32 pairs of tumor and adjacent non-tumor liver tissues., Results: sFGL2 levels were elevated in HBV patients compared to healthy controls (P <  0.0001). In the patient group, sFGL2 levels were increased in AHB compared to CHB patients (P = 0.017). sFGL2 levels were higher in LC patients compared to those without LC (P = 0.006) and were increased according to the development of cirrhosis as staged by Child-Pugh scores (P = 0.024). Similarly, HCC patients had increased sFGL2 levels compared to CHB patients (P = 0.033) and FGL2 mRNA was up-regulated in tumor tissues compared to adjacent non-tumor tissues (P = 0.043). In addition, sFGL2 levels were positively correlated with HBV-DNA loads and AST (Spearman's rho = 0.21, 0.25 and P = 0.006, 0.023, respectively), but reversely correlated with platelet counts and albumin levels (Spearman's rho = - 0.27, - 0.24 and P = 0.014, 0.033, respectively)., Conclusions: sFGL2 levels are induced by HBV infection and correlated with the progression and clinical outcome of HBV-related liver diseases. Thus, sFGL2 may serve as a potential indicator for HBV-related liver diseases.

Background: Circulating microRNAs (miRNA) are biomarkers for several neoplastic diseases, including hepatocellular carcinoma (HCC). We performed a literature search, followed by experimental screening and validation in order to establish a miRNA panel in combination with the assessment of alpha-fetoprotein (AFP) levels and to evaluate its performance in HCC diagnostics., Methods: Expression of miRNAs was quantified by quantitative PCR (qPCR) in 406 serum samples from 118 Vietnamese patients with hepatitis B (HBV)-related HCC, 69 patients with HBV-related liver cirrhosis (LC), 100 chronic hepatitis B (CHB) patients and 119 healthy controls (HC)., Results: Three miRNAs (mir-21, mir-122, mir-192) were expressed differentially among the studied subgroups and positively correlated with AFP levels. The individual miRNAs mir-21, mir-122, mir192 or the triplex miRNA panel showed high diagnostic accuracy for HCC (HCC vs. CHB, AUC = 0.906; HCC vs. CHB+LC, AUC = 0.81; HCC vs. CHB+LC+HC, AUC = 0.854). When AFP levels were ≤20ng/ml, the triplex miRNA panel still was accurate in distinguishing HCC from the other conditions (CHB, AUC = 0.922; CHB+LC, AUC = 0.836; CHB+LC+HC, AUC = 0.862). When AFP levels were used in combination with the triplex miRNA panel, the diagnostic performance was significantly improved in discriminating HCC from the other groups (LC, AUC = 0.887; CHB, AUC = 0.948; CHB+LC, AUC = 0.887)., Conclusions: The three miRNAs mir-21, mir-122, mir-192, together with AFP, are biomarkers that may be applied to improve diagnostics of HCC in HBV patients, especially in HBV-related LC patients with normal AFP levels or HCC patients with small tumor sizes.

Aim: To investigate possible effects of IRF5 polymorphisms in the 3' UTR region of the IFR5 locus on susceptibility to hepatitis B virus (HBV) infection and progression of liver diseases among clinically classified Vietnamese patients., Methods: Four IFR5 SNPs (rs13242262A/T, rs77416878C/T, rs10488630A/G, and rs2280714T/C) were genotyped in clinically classified HBV patients [chronic hepatitis B (CHB). n = 99; liver cirrhosis (LC), n = 131; hepatocellular carcinoma (HCC), n = 149] and in 242 healthy controls by direct sequencing and TaqMan real-time PCR assays., Results: Comparing patients and controls, no significant association was observed for the four IFR5 variants. However, the alleles rs13242262T and rs10488630G contributed to an increased risk of liver cirrhosis (LC vs CHB: OR = 1.5, 95%CI: 1.1-2.3, adjusted P = 0.04; LC vs CHB: OR = 1.7, 95%CI: 1.1-2.6, adjusted P = 0.019). Haplotype IRF5*TCGT constructed from 4 SNPs was observed frequently in LC compared to CHB patients (OR = 2.1, 95%CI: 1.2-3.3, adjusted P = 0.008). Haplotype IRF5*TCAT occurred rather among CHB patients than in the other HBV patient groups (LC vs CHB: OR = 0.4, 95%CI: 0.2-0.8, adjusted P = 0.03; HCC vs CHB: OR = 0.3, 95%CI: 0.15-0.7, adjusted P = 0.003). The IRF5*TCAT haplotype was also associated with increased levels of ALT, AST and bilirubin., Conclusion: Our study shows that IFR5 variants may contribute as a host factor in determining the pathogenesis in chronic HBV infections., Competing Interests: Conflict-of-interest statement: All authors have no conflicts of interest to declare.

This study investigates the association of Interferon-stimulated gene 15 (ISG15) polymorphisms, ISG15 serum levels and expression with HBV-related liver diseases. The ISG15 promoter and the two exons of the gene were screened for polymorphisms in 766 HBV-infected patients and in 223 controls. Soluble ISG15 levels were measured by ELISA. ISG15 mRNA expression was quantified by qRT-PCR in 36 tumor and adjacent non-tumor tissues. The exon 2 allele rs1921A was found associated with decreased progression of HBV-related liver diseases (LC vs. CHB: OR = 0.6, 95%CI = 0.4-0.8, adjusted P = 0.003; HCC vs. CHB: OR = 0.6, 95%CI = 0.4-0.9, adjusted P = 0.005). The rs1921AA genotype was associated with low levels of AST, ALT and total bilirubin, but with high prothrombin levels (P < 0.05). ISG15 serum levels were higher among HBV patients compared to controls (P < 0.0001) and positively associated with HBV-related liver diseases, with highest levels among LC patients. ISG15 levels were correlated with HBV-DNA loads (P = 0.001). In non-tumor tissues from HCC patients, ISG15 mRNA expression was increased in HBV compared to non-HBV infection (P = 0.016). The ISG15 rs1921 variant and ISG15 expression are associated with HBV-related liver diseases. Taken together, ISG15 appears to be a proviral factor involved in HBV replication and triggering progression of HBV-related liver diseases.

Background: The human major histocompatibility complex class I polypeptide-related sequence B (MICB) is a protein that modulates the NK and T cell activation through the NKG2D receptor and is related to several diseases including cancer., Methods: The study investigated the prognostic role of soluble MICB (sMICB) protein in the progression of HBV-related liver diseases and to HBV-related HCC treatment. The sMICB serum levels were measured in 266 chronic HBV-infected Vietnamese patients and in healthy controls, and correlated with clinical and laboratory parameters and with therapeutic interventions for HBV-related HCC., Results: Significant differences in both clinical and laboratory parameters were observed among the patient groups with different stages of hepatitis. The platelet counts were significantly decreased with disease progression (P < 0.001). The sMICB serum levels were significantly increased in HBV patients compared to healthy controls (P < 0.0001). Among the patients with different stages of hepatitis, asymptomatic individuals (ASYM) revealed higher sMICB serum levels while liver cirrhosis (LC) patients revealed lower sMICB serum levels (P < 0.0001) compared to other patient groups. Notably, the sMICB serum levels were decreased in treated HCC patient group compared to not-treated HCC patient group (P = 0.05). Additionally, the sMICB levels were significantly correlated with platelet counts in ASYM and HCC patients (r = -0.37, P = 0.009; and r = 0.22, P = 0.025, respectively)., Conclusions: Our results demonstrate a potential role of sMICB serum levels and platelet counts during immune response to the HBV infection, liver disease progression and response to the HCC treatment.

Background and Aims: This study examined the efficacy of arabinoxylan rice bran (MGN-3) in conjunction with an interventional therapy (IT) for the treatment of hepatocellular carcinoma patients., Patients and Methods: A total of sixty-eight patients with hepatocellular carcinoma (stages I and II) participated in the study. Patients were randomized to receive IT (30 patients, control group) or IT+MGN-3 (38 patients), and randomly divided into two groups using a computer-generated randomization list. Patients and investigators were blinded. IT included transarterial oily chemoembolization (TOCE) or a combination of TOCE and percutaneous ethanol injection treatment (PEIT)., Results: Patients in the IT+MGN-3 group showed: (i) lower recurrence of the disease, 31.6% (12/38), as compared to 46.7% (14/30) for the control; (ii) higher survival after the second year, 35%, as compared to 6.7% for the control; (iii) significantly lower alpha-fetoprotein level, a 38% decrease (p = 0.0001), as compared to baseline value, while the control showed no significant change; and (iv) a significant decrease in tumor volume, in contrast to the control, which showed no significant change. When the results were analyzed according to each IT modality, MGN-3+IT sub-groups displayed a greater response to treatment, in every aspect examined, than the IT sub-groups alone. However, the patients in the MGN-3+TOCE+PEIT sub-group demonstrated greater reduction in AFP levels and longer survival time than the MGN-3+TOCE sub-group., Conclusion: MGN-3 in conjunction with IT may be useful for the treatment of hepatocellular carcinoma and warrants further investigation in multiple clinical trials.