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Dysregulation of expression of functional genes and pathways plays critical roles in the etiology and progression of hepatocellular carcinoma (HCC). Next generation-based RNA sequencing (RNA-seq) offers unparalleled power to comprehensively characterize HCC at the whole transcriptome level. In this study, 17 fresh-frozen HCC samples with paired non-neoplastic liver tissue from Caucasian patients undergoing liver resection or transplantation were used for RNA-seq analysis. Pairwise differential expression analysis of the RNA-seq data was performed to identify genes, pathways, and functional terms differentially regulated in HCC versus normal tissues. At a false discovery rate (FDR) of 0.10, 13% (n = 4335) of transcripts were up-regulated and 19% (n = 6454) of transcripts were down-regulated in HCC versus non-neoplastic tissue. Eighty-five Kyoto Encyclopedia of Genes and Genomes pathways were differentially regulated (FDR, <0.10), with almost all pathways (n = 83) being up-regulated in HCC versus non-neoplastic tissue. Among the top up-regulated pathways was oxidative phosphorylation (hsa00190; FDR, 1.12E-15), which was confirmed by Database for Annotation, Visualization, and Integrated Discovery (DAVID) gene set enrichment analysis. Consistent with potential oxidative stress due to activated oxidative phosphorylation, DNA damage-related signals (e.g., the up-regulated hsa03420 nucleotide excision repair [FDR, 1.14E-04] and hsa03410 base excision repair [FDR, 2.71E-04] pathways) were observed. Among down-regulated genes (FDR, <0.10), functional terms related to cellular structures (e.g., cell membrane [FDR, 3.05E-21] and cell junction [FDR, 2.41E-07], were highly enriched, suggesting compromised formation of cellular structure in HCC at the transcriptome level. Interestingly, the olfactory transduction (hsa04740; FDR, 1.53E-07) pathway was observed to be down-regulated in HCC versus non-neoplastic tissue, suggesting impaired liver chemosensory functions in HCC. Our findings suggest oxidative phosphorylation and the associated DNA damage may be the major driving pathologic feature in HCC., (© 2022 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Most studies about serpin peptidase inhibitor, clade A member 3 (SERPINA3) has been limited to its inhibitory functions and mechanisms. Herein, we report a novel role of SERPINA3 in transcriptional regulation of HCC progression-related genes. Among 19 selected genes through HCC cell isolation system based on telomere length, microarray analyses, and cell-based studies, SERPINA3 was the strongest determinant of increases in telomere length, HCC cell proliferation, survival, migration, and invasion. We also found that SERPINA3 strongly interacted with heterogeneous nuclear ribonucleoprotein K (HNRNP-K) under H 2 O 2 exposure, and the oxidation-elicited SERPINA3-HNRNP-K complex enhanced the promoter activities and transcript levels of a telomere-relating gene (POT1) and HCC-promoting genes (UHRF1 and HIST2H2BE). Intriguingly, the inhibition of SERPINA3 oxidation rendered the transcriptional activity of the SERPINA3-HNRNP-K complex suppressed. Moreover, the co-immunoprecipitated HNRNP-K with SERPINA3 quantitatively correlated with not only the level of SERPINA3 oxidation but also the level of POT1, UHRF1, and HIST2H2BE transcripts and telomere length in HCC tissues. Therefore, the upregulated transcriptional activity of HNRNP-K mediated by SERPINA3 promotes HCC cell survival and proliferation and could be an indicator of poor prognosis for HCC patients., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Class I phosphoinositide 3-kinase (PI3K) signaling is a major pathway in human cancer development and progression. Among the four PI3K isoforms, PI3Kα and PI3Kβ are ubiquitously expressed, whereas PI3Kγ and PI3Kδ are found primarily in leukocytes. Until now, PI3K targeting in solid tumors has focused on inhibiting PI3Kα-mediated and PI3Kβ-mediated cancer cell-intrinsic PI3K activity. The role of PI3Kδ in solid tumors is unknown. Here, we evaluated the effects of PI3Kδ using established hepatocellular carcinoma (HCC) cells, malignant hepatocytes derived from patients with advanced HCC, murine models, and HCC tissues using RNA sequencing, quantitative PCR, immunoblotting, immunofluorescence, microarray, liquid chromatography-tandem mass spectrometry, and kinase assay. We established a chemical carcinogenesis model of liver malignancy that reflects the malignant phenotype and the in vivo environment of advanced HCC. In this in vivo advanced HCC-mimic system using HCC cells treated with hydrogen peroxide (H 2 O 2 ), we showed that H 2 O 2 selectively increases PI3Kδ activity while decreasing that of other class I PI3Ks. Blocking PI3Kδ activity with a PI3Kδ inhibitor or small interfering RNA-mediated PI3Kδ gene silencing inhibited HCC-cell proliferation and dampened key features of malignant HCC, including the up-regulation of telomerase reverse transcriptase (TERT). Mechanistically, H 2 O 2 induced oxidative modification of the serpin peptidase inhibitor, serpin peptidase inhibitor (SERPINA3), blocking its ubiquitin-dependent degradation and enhancing its activity as a transcriptional activator of PI3Kδ and TERT. High PI3Kδ levels in HCC were found to correlate with poor survival rates, with human advanced HCC showing positive correlations between the protein levels of oxidized SERPINA3, PI3Kδ, and TERT. Thus, PI3Kδ plays significant roles in malignant liver tumors. Conclusion: Our data identify PI3Kδ inhibition, recently approved for the treatment of human B-cell malignancies, as a potential treatment for HCC., (© 2018 by the American Association for the Study of Liver Diseases.)

Objective: To evaluate the relationship between donor sex and hepatocellular carcinoma (HCC) recurrence after living donor liver transplantation., Background: HCC shows a male predominance in incidence and recurrence after tumor resection due to sex differences in hepatic sex hormone receptors. There have been no studies evaluating the importance of donor sex on post-transplant HCC recurrence., Methods: Of 384 recipients of livers, from living donors, for HCC: 104/120 who received grafts from female donors were matched with 246/264 who received grafts from male donors using propensity score matching, with an unfixed matching ratio based on factors like tumor biology. Survival analysis was performed with death as a competing risk event. The primary outcome was overall HCC recurrence., Results: The median follow-up time was 39 months. Before matching, recurrence probability at 1/2/5 years after transplantation was 6.1/9.7/12.7% in recipients with female donors and 11.7/19.2/25.3% in recipients with male donors. Recurrence risk was significantly higher with male donors in univariable analysis (hazard ratio [HR] = 2.04 [1.15-3.60], P = 0.014) and multivariable analysis (HR=2.10 [1.20-3.67], P = 0.018). In the matched analysis, recurrence risk was also higher with male donors (HR=1.92 [1.05-3.52], P = 0.034): both in intrahepatic recurrence (HR=1.92 [1.05-3.51], P = 0.034) and extrahepatic recurrence (HR=1.93 [1.05-3.52], P = 0.033). Multivariable analysis confirmed the significance of donor sex (HR=2.08 [1.11-3.91], P = 0.023). Interestingly, the significance was lost when donor age was >40 years. Two external cohorts validated the significance of donor sex., Conclusions: Donor sex appears to be an important graft factor modulating HCC recurrence after living donor liver transplantation.

Dyskerin pseudouridine synthase 1 (DKC1) is a conserved gene encoding the RNA-binding protein dyskerin, which is an essential component of the telomerase holoenzyme. DKC1 up-regulation is frequently observed in many different human cancers including hepatocellular carcinoma (HCC); however, its regulatory mechanisms remain unclear. Thus, we investigated the regulatory mechanism of DKC1 in HCC progression. We found that protein-disulfide isomerase-associated 3 (PDIA3) interacted with the DKC1 regulatory DNA in HCC cells but not in HCC cells with elevated reactive oxygen species (ROS) levels, using liquid chromatographic-tandem mass spectrometric analysis after isolating the DKC1 regulatory region binding proteins. PDIA3 repressed DKC1 expression in HCC cells by recognizing the G-quadruplex DNA at the DKC1 location. However, oxidative modification of PDIA3 induced by ROS redistributed this protein into the cytosolic regions, which stimulated DKC1 expression. We also identified Met338 in PDIA3 as the oxidatively modified residue and validated the effect of oxidative modification using an ectopic expression system, a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 knock-in system, and a xenograft mouse model. We observed that oxidatively modified PDIA3 promoted DKC1-mediated malignancy and survival of HCC cells in vitro and in vivo. HCC tissues showed a positive association with ROS, cytoplasmic PDIA3, and nuclear DKC1 levels. HCC patients with high PDIA3 protein and DKC1 mRNA levels also displayed reduced recurrence-free survival rates. Cumulatively, the results showed that cytoplasmic PDIA3 activity could be essential in raising DKC1 expression in HCC progression and predicting poor prognoses in HCC patients. Conclusion: Our study indicates that the elevated ROS levels in HCC modulate cytoplasmic PDIA3 levels, resulting in HCC cell survival through DKC1 up-regulation., (© 2018 by the American Association for the Study of Liver Diseases.)

Telomeres protect chromosomal ends from deterioration and have been shown to be susceptible to shortening by reactive oxygen species (ROS)-induced damage. ROS levels increase during the progression from early to advanced hepatocellular carcinoma (HCC). An independent study found that the telomeres in most HCC tissues lengthened during carcinogenic advancement. Activated telomerase has been hypothesized to elongate telomeres during the progression of malignant HCC, but it remains unclear which signaling pathway is necessary for telomerase activation in HCC. Here, we showed using cell lines derived from human HCC that H 2 O 2 , which is a major component of ROS in living organisms, elongates telomeres by increasing telomerase activity through protein kinase B (AKT) activation. The AKT inhibitor, perifosine, decreased telomere length, cellular viability, and H 2 O 2 -mediated migration and invasion capacity in HCC cells while also inhibiting AKT activation, telomere maintenance, and tumor growth in nude mice. Advanced HCC tissues showed a positive correlation among ROS levels, phosphorylated AKT (pAKT) levels, and telomere length. Furthermore, patients with HCC tumors that have high ROS levels and long telomeres displayed poorer survival rates. These data demonstrate the significant utilities of ROS levels, pAKT levels, and telomere length for predicting a poor prognosis in patients with HCC. Taken together, AKT activation could be essential for telomere maintenance in advanced HCC tumors as well as being an important contributor to malignant HCC progression., Conclusion: We showed that H 2 O 2 contributes to telomere elongation through AKT activation in advanced HCC, suggesting that an AKT inhibitor such as perifosine may be useful for treating patients with malignant HCC. (Hepatology 2018;67:1378-1391)., (© 2017 by the American Association for the Study of Liver Diseases.)

A common single-nucleotide polymorphism in the telomerase reverse transcriptase (TERT) promoter, rs2853669 influences patient survival rates and the risk of developing cancer. Recently, several lines of evidence suggest that the rs2853669 suppresses TERT promoter mutation-mediated TERT expression levels and cancer mortality as well as recurrence rates. However, no reports are available on the impact of rs2853669 on TERT expression in hepatocellular carcinoma (HCC) and its association with patient survival. Here, we found that HCC-related overall and recurrence-free survival rates were not associated with TERT promoter mutation individually, but rs2853669 and the TERT promoter mutation in combination were associated with poor survival rates. TERT mRNA expression and telomere fluorescence levels were greater in patients with HCC who had both the combination. The combination caused TERT promoter methylation through regulating the binding of DNA methyltransferase 1 and histone deacetylase 1 to the TERT promoter in HCC cell lines. The TERT expression level was significantly higher in HCC tumor with a methylated promoter than in that with an unmethylated promoter. In conclusion, we demonstrate a substantial role for the rs2853669 in HCC with TERT promoter mutation, which suggests that the combination of the rs2853669 and the mutation indicate poor prognoses in liver cancer.

Hepatocellular carcinoma downregulated 1 (HEPN1), a cell growth arrest- and apoptosis-related gene, is suppressed in hepatocellular carcinoma (HCC). However, transcriptional control of HEPN1 has not been characterized. Here, we show that exposure to reactive oxygen species (ROS) leads to upregulation of the mRNA expression of HEPN1 in HCC cell lines. Mechanistically, ROS increase production of an alternately spliced form of X-box binding protein 1 (XBP1s) and XBP1s increases HEPN1 expression by binding to the HEPN1 promoter, thereby acting as a transcriptional activator. Finally, HEPN1 overexpression increases the expression of p53, p21, and Bax, all of which are ROS-upregulated proteins., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Notch1 and reactive oxygen species (ROS) modulate important pathways associated with tumor development and progression. Notably, Notch1 expression is upregulated in 41.8% of hepatocellular carcinoma (HCC) patients and ROS levels increases as HCC progresses from Grade I to Grade III. It has been established that Notch1 and ROS modulate Snail expression in malignant tumors; however, the mechanism regulating Snail protein expression is not yet known. In this study, we observed that Notch1 and ROS cooperatively increase the levels of Snail protein in Huh7 (hepatoma) cells. On its own, signaling through Notch1 increases transcription of Snail without changing protein levels. In contrast, the combined activation of the Notch1 and ROS-induced phosphoinositide 3-kinase/Akt (PI3K/Akt) signaling pathways resulted in the high expression of Snail protein. This increase in Snail expression was associated with increased Huh7 cells invasiveness. Furthermore, we observed that correlation between Snail and Notch1 expression was the strongest in advanced grade HCC tissue. In conclusion, Notch1 and ROS-induced PI3K/Akt signals cooperatively increase Snail expression and may induce malignancy in HCC.

Invasion, the representative feature of malignant tumors, leads to an increase in mortality. The malignant liver tumor - hepatocellular carcinoma (HCC) - has an enhanced invasive capacity that results in increased patient mortality. Moreover, this enhanced invasive capacity is due to the up-regulation of invasion promoters such as zinc finger protein SNAI1 (Snail) and matrix metalloproteinases (MMPs), and the down-regulation of invasion suppressor molecules such as E-cadherin. Telomerase reverse transcriptase (TERT), which encodes the catalytic subunit of telomerase, is highly expressed in a variety of invasive cancers, including HCC. Telomerase activation induces telomere elongation, thereby leading to cell immortalization during malignant tumor progression. However, the relationship between telomere length and invasion is yet to be experimentally corroborated. In this paper, we revealed that invasive HCC cells passing through the Matrigel display significantly longer telomeres than non-invasive HCC cells. Moreover, we established a method that can distinguish and sort cells containing long telomeres and short telomeres. Using this system, we observed that the HCC cells containing long telomeres had a high-level expression of invasion-promoting genes and a low-level expression of invasion-suppressing E-cadherin. Furthermore, HCC cells containing long telomeres exhibited a higher invasive capacity than HCC cells containing short telomeres. Taken together, our findings suggest that long telomeres are positively associated with the invasive capacity of HCC cells and may be a potent target for malignant liver cancer treatment., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background/aims: We aimed to determine the association between the co-expression patterns of Notch1, Snail, and p53 proteins (NSP) and the postoperative prognosis of hepatocellular carcinoma (HCC)., Methods: The immunoblot data for molecular expression (147 HCC/corresponding non-HCC tissues and 15 dysplastic nodules) and the sequencing data for p53 mutations (110 HCCs) were obtained from our previous study. Data analyses were restricted to cases with HCC differentiation grade III (n=47), due to its high p53 mutation rate., Results: Nineteen of the 47 patients (40.4%) -comprising 12 in the liver and 7 in distant organs-had relapsed at 1-2 years after surgery. There was no relationship between p53 mutation and postoperative recurrence in the grade III HCCs. Seven (87.5%) of the eight relapsed cases with Notch1, Snail, and p53 (wild) co-expression experienced recurrence only within the liver, and all tumors were smaller than 5 cm in diameter. Extrahepatic relapse occurred mostly in HCC patients with tumors larger than 5 cm in diameter, without any deviation in the NSP pattern., Conclusions: The results of this preliminary study suggest that the co-expression of Notch1, Snail, and p53 (wild) is not inferior to the patterns with p53 mutation as an indicator of postoperative recurrence of grade III HCC.

Objective: Salvage liver transplantation (LT) has been proposed for patients with a small hepatocellular carcinoma (HCC) and preserved liver function. Few reports have been issued on salvage LT in a living-donor (LD) LT setting. Therefore, we performed this study to evaluate differences in tumor invasiveness and other risk factors on survival after salvage versus primary LDLT., Methods: Between September 1996 and December 2008, 324 patients with HCC underwent LT. We excluded 138 patient from the analysis, leaving 186 HCC patients for analysis, including 17 (9.1%) who had undergone earlier resection, the salvage LDLT cohort. The other 169 patients underwent primary LDLT., Results: Intrahepatic metastasis, Edmonson-Steiner histologic grade, microscopic vascular invasion, and preoperative serum alpha-fetoprotein levels significantly influenced tumor recurrence. Microscopic vascular invasion, intrahepatic metastasis, Edmonson-Steiner histologic grade, and treatment by salvage LDLT were significantly associated with poor patient survival univariate analysis. However, only microscopic vascular invasion was significant on multivariate analysis. The treatment modality (primary or salvage LDLT) was not observed to affect overall or disease-free survival significantly on multivariate analysis. Disease-free survival was significantly better in the primary than in the salvage LDLT group. Furthermore, patients in the primary LDLT group tended to show better survival. However, when stratified by the presence or absence of microscopic vascular invasion, no significant group difference was found for overall or disease-free survival among those without versus with microscopic vascular invasion., Conclusions: Five-year overall survival after primary versus salvage LDLT were similar when differences in tumor pathologic features, such as microscopic vascular invasion, were taken into account. Multivariate analysis showed that the treatment itself was not a significant prognostic factor for survival., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Hepatocellular carcinoma (HCC) is a common, highly invasive malignant tumor associated with a high mortality rate. We previously reported that the aberrant expression of Snail via activation of reactive oxygen species contributes to the invasive property of HCC, in part by downregulation of E-cadherin through both transcriptional repression and epigenetic modification of the E-cadherin promoter. Having demonstrated the ability of Snail to bind and recruit histone deacetylase 1 and DNA methyltransferase 1 in this context, we set out to look for other interactions that could affect its ability to promote oncogenic transformation and cancer cell invasion., Results: Using cells that stably expressed Snail, we characterized Snail protein interactors by tandem affinity purification and mass spectrometry. Immunoprecipitation and subcellular colocalization studies were performed to confirm our identification of the Notch1 intracellular domain (NICD) as a novel Snail-binding partner. NICD interaction with Snail was found to induce ubiquitination and MDM2-dependent degradation of Snail. Interestingly, NICD inhibited Snail-dependent invasive properties in both HCC cells and mouse embryonic fibroblasts., Conclusions: Our study demonstrates that NICD can oppose Snail-dependent HCC cell invasion by binding and inducing proteolytic degradation of Snail. Although Notch signaling and Snail are both widely considered tumor-promoting factors, our findings indicate that the individual oncogenic contribution of Notch1 and Snail in malignant systems should be interpreted carefully, particularly when they are conjointly expressed.

Background/aims: Combination treatment consisting of hepatic arterial infusion chemotherapy with epirubicin and cisplatin (HAIC-EC) and systemic infusion of low-dose 5-fluorouracil (5-FU) are sometimes effective against advanced hepatocellular carcinoma (HCC). However, there is no effective treatment for advanced HCCs with arterioportal shunts (APS) or arteriovenous shunts (AVS)., Methods: We investigated a response and adverse events of a new combination protocol of repeated HAIC-EC and percutaneous intratumoral injection chemotherapy with a mixture of recombinant interferon-gamma (IFN-γ) and 5-FU (PIC-IF) in patients with far-advanced HCCs with large APSs or AVSs., Results: There was a complete response (CR) for the large vascular shunts in all three patients and for all tumor burdens in two patients. Significant side effects were flu-like symptoms (grade 2) and bone marrow suppression (grade 2 or 3) after each cycle, but these were well-tolerated., Conclusions: These results suggest that the combination of HAIC-EC and PIC-IF is a new and promising approach for advanced HCC accompanied by a large APS or AVS.

Background & Aims: The concept of multistep hepatocarcinogenesis has been well-established, and an accumulation of methylating events has recently been demonstrated; however, the methylation status of low-grade dysplastic nodules (LGDN), high-grade dysplastic nodules (HGDN), and the recently introduced early hepatocellular carcinoma (eHCC) in hepatitis B virus (HBV)-related hepatocarcinogenesis has not yet been studied., Methods: One hundred thirty-three DNA samples (45 cirrhotic nodules, 29 LGDNs, 13 HGDNs, 14 eHCCs, and 32 progressed HCCs (pHCCs)) from HBV-infected resected livers were subjected to MethyLight analysis for nine CpG island loci (APC, RASSF1A, SOCS1, P16, COX2, SPRY2, PTEN, GNMT, and ERK), and COX2, RASSF1A, and SOCS1 protein expression status was analyzed by immunohistochemistry. The methylation status of each sample was correlated with the clinicopathological features., Results: APC, RASSF1A, and SOCS1 were methylated in 20 (44.4%), 25 (55.6%), and 13 (28.9%) of 45 cirrhosis samples, and APC (p=0.0008) and SOCS1 (p=0.0187) methylation were more frequent in dysplastic nodules and HCCs. APC (p=0.001) and RASSF1A (p=0.019) methylation levels were significantly increased from cirrhosis to LGDN. SOCS1 methylation gradually increased along multistep hepatocarcinogenesis, peaked at eHCC and decreased significantly in pHCCs (p=0.039). By contrast, p16 and COX2 was only methylated in dysplastic nodules and HCCs, with a stepwise increase up to pHCCs. As a whole, the frequency of methylation was highest in eHCCs. A stepwise decrease in COX2, RASSF1A, and SOCS1 protein expression was demonstrated., Conclusions: A general stepwise increase in methylating events is seen during HBV-related multistep hepatocarcinogenesis, and epigenetic changes may occur predominantly in the earlier stages of HCC development., (Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Unlabelled: The tumor suppressor p53 is a key prognostic factor in hepatocellular carcinoma (HCC), yet only 35% of grade III tumors exhibit mutation of p53. Several other pathways have been implicated in HCC and, among these, the role of the Notch1/Snail pathway remains unclear. Therefore, we investigated the expression of p53, Notch1, and Snail proteins in HCC with regard to both clinical grade and p53 mutational status. Immunoblotting for p53 revealed that, whereas in many tumors increased p53 was a result of p53 mutation, wildtype p53 (p53WT) expression was also frequently elevated in HCCs. Coordinated evaluation of p53, Notch1, and Snail expression suggests that grade III HCC can be subdivided based on the expression of these three proteins. We found that Notch1 expression in HCC tissues and cell lines is differentially affected by p53WT and mutant p53 (p53Mut). Notch1 expression was correlated with p53 expression in cells expressing p53WT, but was not elevated in p53Mut-expressing cells. Virally mediated expression or silencing of p53WT or p53Mut confirmed that p53WT overexpression causes Notch1 up-regulation in HCC. Surprisingly, the consequence of Notch1 overexpression for the proliferative and invasive capacity of HCC cells depends on both the p53 mutational status and activation of the Snail pathway., Conclusion: In the presence of p53WT, Snail/Notch1 activation increased the invasiveness of HCC cells. In contrast, in the absence of p53WT, Notch1 decreased the invasiveness of HCC. Taken together, these findings shed new light on the complex role of the Notch1/Snail axis in HCC and provide a framework for further classifying HCC based on the expression and mutational status of p53 and the expression of Notch1 and Snail., (American Association for the Study of Liver Diseases.)

The tumor suppressor protein p53 is a key regulator of cell cycle arrest and apoptosis. Snail protein regulates cancer-associated malignancies. However, the relationship between p53 and Snail proteins in hepatocellular carcinoma (HCC) has not been completely understood. To determine whether Snail and p53 contribute to hepatocarcinogenesis, we analyzed the expression of Snail proteins in p53-overexpressing HCC cells. We found that p53 wild-type (WT) induced the degradation of Snail protein via murine double minute 2-mediated ubiquitination, whereas p53 mutant did not induce Snail degradation. As we expected, only p53WT induced endogenous Snail protein degradation and inhibited tumor cell invasion. These findings contribute to a better understanding of the role of p53 mutation and Snail overexpression as a late event in hepatocarcinogenesis., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Patients with unresectable, beyond Milan criteria, hepatocellular carcinoma (HCC) invariably undergo palliative transarterial chemoembolization (TACE). The aim of this study was to compare the outcomes of conventional TACE versus liver transplantation (LT) in unresectable (beyond Milan criteria) HCC. Twelve patients underwent LT and 86 TACE for unresectable, beyond Milan criteria HCC. The inclusion criteria were a single tumor60 years, vascular invasion, or extrahepatic spread. Survival rates were calculated using the Kaplan-Meier method. Multivariate analysis showed that TACE was a prognostic factor for survival (hazard ratio, 16.66, P=.000). The LT group showed significantly better survival than the TACE cohort. Two cases (16.7%) in the LT group recurred at a median time of 13.5 months. Survival rates at 1, 3, and 5 years were 100%, 88.9%, and 76.2% in the LT group, and 85.6%, 45.6%, and 21.4% in the TACE group, respectively. Patients with unresectable, beyond Milan criteria HCC should be given the option to receive LDLT, because LT offers a significantly better likelihood of survival than TACE., (Copyright (c) 2010. Published by Elsevier Inc.)

Background/aims: The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) has been implicated in HCV-induced liver pathogenesis. Wnt/beta-catenin signaling has also been involved in tumorigenesis. To elucidate the molecular mechanism of HCV pathogenesis, we examined the potential effects of HCV NS5A protein on Wnt/beta-catenin signal transduction cascades., Methods: The effects of NS5A protein on beta-catenin signaling cascades in hepatic cells were investigated by luciferase reporter gene assay, confocal microscopy, immunoprecipitation assay, and immunoblot analysis., Results: beta-Catenin-mediated transcriptional activity is elevated by NS5A protein, in the context of HCV replication, and by infection of cell culture-produced HCV. NS5A protein directly interacts with endogenous beta-catenin and colocalizes with beta-catenin in the cytoplasm. NS5A protein inactivates glycogen synthase kinase 3beta and increases subsequent accumulation of beta-catenin in HepG2 cells. beta-Catenin was also accumulated in HCV patients' liver tissues. In addition, the accumulation of beta-catenin in HCV replicon cells requires both activation of phosphatidylinositol 3-kinase and inactivation of GSK3beta., Conclusions: NS5A activates beta-catenin signaling cascades through increasing the stability of beta-catenin. This modulation is accomplished by the protein interplay between viral and cellular signaling transducer. These data suggest that NS5A protein may directly be involved in Wnt/beta-catenin-mediated liver pathogenesis.

Background & Aims: In addition to genetic alterations, epigenetic changes underlie tumor progression and metastasis. Promoter methylation can silence tumor suppressor genes, and reactive oxygen species (ROS) promote DNA damage, although the relationship between ROS and epigenetic changes in cancer cells is not clear. We sought to determine whether ROS promote hypermethylation of the promoter region of E-cadherin, a regulator of the epithelial-to-mesenchymal transition, in hepatocellular carcinoma (HCC) cells., Methods: HCC cells were exposed to H(2)O(2) or stably transfected to express Snail, a transcription factor that down-regulates E-cadherin expression. E-cadherin and Snail expression levels were examined by real-time reverse-transcriptase polymerase chain reaction and immunoblot analyses. The methylation status of E-cadherin was examined by methyl-specific polymerase chain reaction, bisulfite sequencing, and chromatin immunoprecipitation. The interactions between Snail, histone deacetylase 1, and DNA methyltransferase 1 were assessed by immunoprecipitation/immunoblot and immunofluorescence analyses. ROS-induced stress, E-cadherin expression, Snail expression, and E-cadherin promoter methylation were confirmed in HCC tissues by immunoblot, immunohistochemistry, and methyl-specific polymerase chain reaction analyses., Results: We demonstrated that ROS induce hypermethylation of the E-cadherin promoter by increasing Snail expression. Snail induced DNA methylation of the E-cadherin promoter by recruiting histone deacetylase 1 and DNA methyltransferase 1. In human HCC tissues, we observed a correlation among ROS induction, E-cadherin down-regulation, Snail up-regulation, and E-cadherin promoter methylation., Conclusions: These findings provide novel mechanistic insights into epigenetic modulations induced by ROS in the process of carcinogenesis. They are potentially relevant to understanding the activity of ROS in silencing various tumor suppressor genes and in subsequent tumor progression and metastasis.

In December 2006 the allocation of livers from deceased donors in Germany and several other Eurotransplant countries was reset. The previous allocation system relied on CTP score to assess the need of transplantation, but it also assigned to waiting time a prominent role in prioritization. That system was replaced by the primarily urgency-oriented model of end-stage liver disease (MELD) allocation system. First experience with this classification in the U.S.A. shows that MELD scores are able to identify the urgency of liver transplantation correctly in most types of liver disease. Due to the MELD-based allocation, the growing waiting time and waiting-list mortality could be counteracted. At the same time it became evident however that MELD scores do not reflect mortality on the waiting list or thus the urgency for all types of liver diseases. Therefore the new allocation system introduced in the Eurotransplant countries contains standardized and flexible exceptions for these diseases. In addition the new allocation rules were created as a learning system. Repeated "fine tuning" of the allocation process based on continuous monitoring of daily allocation practice and clinical studies aim at just and effective distribution of the precious and limited supply of donor organs.

Nuclear factor (NF)-kappaB is important for immune responses and cell survival; however, abnormal activation of NF-kappaB is linked with many types of diseases, including hepatocellular carcinoma (HCC). Our previous report indicated that hepatitis B virus (HBV) induces NF-kappaB activation through NF-kappaB-inducing kinase (NIK), and this can be blocked specifically by interferon (IFN)-gamma. In the present study, we report that HBV expression in HCC cell lines induces drug resistance against 5-fluorouracil (5-FU). This drug resistance was abolished by inhibition of NF-kappaB activation through small interfering RNA-mediated NIK 'knockdown' and IFN-gamma treatment. In addition to the reduced NF-kappaB activation and drug resistance, the upregulated growth arrest- and DNA damage-inducible protein 45beta (Gadd45beta) in HBV-expressing HCC cell lines was downregulated by the small interfering RNA-mediated NIK knockdown and IFN-gamma treatment. The overexpression of Gadd45beta in HCC cell lines also induces drug resistance against 5-FU. Based on our data, we suggest that IFN-gamma treatment might be helpful for chemotherapy in HBV-integrated HCC through inhibition of the NIK-mediated NF-kappaB activation and downregulation of the NF-kappaB target gene Gadd45beta.

Objective: In our multi center trial we compared the potentials of biphasic contrast-enhanced computed tomography (CT) and a novel tissue-specific magnetic resonance imaging (MRI) contrast agent gadoxetic acid disodium in liver lesion characterization., Methods: A total of 176 patients with 252 liver lesions were analyzed. There were 104 malignant and 148 benign lesions. High-field strength (1.0 T or 1.5 T) MR systems with T1-and T2-weighted sequences were used with and without fat suppression. After gadoxetic acid disodium injection, dynamic imaging and hepatocyte phase MR imaging were performed. Biphasic with 150 mg I/kg of body weight (100-200 mL) spiral CT was also performed. Image reading consisted of on-site (by study investigators) and fully blinded off-site (by E.S.P; C.R; and A.S) evaluations. The classification (benign or malignant) and characterization (lesion type) outcomes of both techniques were assessed. All imaging results were verified against a standard of reference., Results: Both on-site and off-site evaluations demonstrated increases in the lesion classification accuracy with gadoxetic acid disodium-enhanced MRI when compared with spiral CT. This improvement was also shown for characterization. Gadoxetic acid disodium was well tolerated., Conclusions: Gadoxetic acid disodium offers a safe and diagnostically powerful tool for the evaluation of patients with focal liver lesions with a reliable assessment of lesion classification and characterization.

Purpose: To evaluate the ability of contrast-enhanced magnetic resonance imaging (MRI) with Gd-EOB-DTPA in comparison with non-enhanced imaging and spiral computed tomography (CT) to provide additional information for classification and characterization of hepatocellular carcinoma., Material and Methods: Forty patients with histopathology-proven hepatocellular carcinoma were selected for this subgroup analysis from a phase-III multicenter study in 235 patients with known or suspected liver lesions. The primary analysis was comparison of the proportion of hepatocellular carcinoma correctly classified and characterized by combined pre-/post-contrast MRI compared with pre-contrast MRI alone or with spiral CT. All images were evaluated on site, and in a blinded reading by three independent readers off site., Results: In the on-site evaluation, the lesions were correctly classified as a malignant tumor with combined MRI in 90.3%, with pre-contrast imaging alone in 82.9% and with spiral CT in 87.8% (n.s.). The proportion of correct characterization (lesion type diagnosis) with combined MRI was 85.4%, 75.6% for pre-contrast imaging, and 77.5% for spiral CT (n.s.), respectively. In the blinded reading, one reader showed a significant increase in the proportion of correctly characterized lesions by 27% (P<0.05). The other two readers showed a reduction in the proportion of correct characterization by 12% and 15%, respectively (n.s.)., Conclusion: With regard to lesion classification, no difference was found between combined pre-/post-contrast MRI and spiral CT. A non-significant trend in favor of Gd-EOB-DTPA-enhanced MRI with regard to characterization of hepatocellular carcinoma was found, although the CT scans were not optimized as the MRI scans.

Purpose: To evaluate the ability of contrast-enhanced MRI with SHU 555 A to provide additional information for characterization of focal liver tumors compared with non-enhanced MRI and multislice spiral CT., Materials and Methods: In a prospective manner the images of 45 patients who underwent multislice spiral CT, unenhanced MRI alone and unenhanced and SHU 555 A-enhanced MRI including dynamic imaging at a field strength of 1.0 T were analyzed in a blinded reading. The readers had to determine on a scale from 1 to 5 whether a tumor was benign or malignant. Furthermore, the readers had to give a definitive diagnosis for each lesion. A true cut needle biopsy served as gold standard against which all imaging procedures were compared., Results: The sensitivity for differentiation malignant vs. benign lesion was 77 % with spiral CT, 72 % with unenhanced MRI and 94 % with SHU 555 A-enhanced MRI, respectively (p < 0.05). The specificity for spiral CT was 73 %, for unenhanced MRI 83 % and for contrast-enhanced MRI 83 %, respectively (n. s.). Compared with the histopathologic results, the correct diagnosis was made with spiral CT in 25/45 (56 %), unenhanced MRI in 16/45 (36 %) and contrast-enhanced MRI in 32/45 (71 %) of the patients (p < 0.05). For the subgroup of patients with liver cirrhosis, the correct diagnosis was established with spiral CT in 16/23 (70 %), unenhanced MRI in 9/23 (39 %) and contrast-enhanced MRI in 19/23 (83 %) of the patients (p < 0.05)., Conclusion: Contrast-enhanced MRI with SHU 555 A has the ability to improve the differential diagnosis of focal liver tumors compared with unenhanced MRI and multislice spiral CT.

Background & Aims: Nuclear factor-kappaB (NF-kappaB) signaling pathway is an important regulating pathway in liver diseases, including hepatocellular carcinoma. In our study, immunohistochemical analysis showed that NF-kappaB-inducing kinase (NIK), an upstream kinase of IkappaB kinases, nuclear localization occurs only in liver tissues obtained from hepatitis B surface antigen (HBsAg)(+) patients but not in tissues from HBsAg(-) patients. The aim of the present study was to identify the inducer of NIK nuclear localization and determine whether the NIK nuclear localization affects the hepatitis B virus (HBV)-mediated NF-kappaB activation., Methods: The experiments were performed on HepG2.2.15 cells and on HepG2 cells transfected with pHBV1.2x, a plasmid encoding all HBV messages, using NF-kappaB-dependent luciferase reporter gene assay, electrophoretic mobility shift assay, immunoblot analysis, and fluorescent microscopy analysis., Results: HBV induced NIK-dependent NF-kappaB activation. However, interferon (IFN)-gamma induced NIK nuclear localization and inhibited NF-kappaB activation in HepG2.2.15 cells and in HepG2 cells transfected with pHBV1.2x. When NIK nuclear localization was inhibited by deletion of nuclear localization signal on NIK, IFN-gamma did not induce the NIK nuclear localization and did not inhibit NF-kappaB activation., Conclusions: IFN-gamma selectively inhibits HBV-mediated NF-kappaB activation. This inhibition is accomplished by NIK nuclear localization, which is a novel mechanism of NF-kappaB inhibition.

Non-steroidal anti-inflammatory drugs (NSAIDs) have anti-proliferative effects and induce apoptosis in colon and other cancers. In the present study, we report that mefenamic acid (MEF), a member of NSAIDs, has an inhibitory effect on a proliferation of liver cancer cells. We used Chang and Huh-7 cells as human liver cancer cells. MEF-treated Huh-7 and Chang cells displayed apoptotic morphological changes and the portion of cells in sub G1 was increased 3-fold and 6-fold, respectively, at a 200 microM concentration. We also show an MEF-enhanced binding of annexin V to cells and an increased activity of caspase-3 to cleave PARP-1 and caspase itself. The inhibitor of caspase-3 blocked PARP-1 cleavage activity and protected against MEF-induced apoptotic cell death. These results indicate that MEF induces apoptosis in human liver cancer cells.

In a 65-year-old female patient, B-mode sonography detected a single focal lesion in the right liver lobe with a diameter < 3 cm. Histopathologic examination revealed a low differentiated hepatocellular carcinoma (HCC; G3). Tumor staging was performed by CT (computed tomography) scan and Resovist MRI (magnetic resonance imaging). Both examinations found a single liver lesion without signs of additional focal hepatic lesions. In addition, phase-inversion sonography in the late phase was performed using the ultrasound contrast agent Levovist. This examination of late-phase Levovist uptake detected more than five additional focal hepatic lesions in the right liver lobe, which were invisible by CT scan and Resovist MRI. This finding of multiloculated HCC was very important to decide on the patient's correct therapy. While liver transplantation is the treatment of choice in single HCC < 3 cm, it is contraindicated in multicentric HCC. In the patient described here, hemihepatectomy of the right liver lobe was performed. The histopathologic examination of the resected liver confirmed the diagnosis of multicentric HCC, which was noninvasively diagnosed only by contrast-enhanced sonography, but not by CT scan or MRI.

Purpose: Hepatocellular carcinoma (HCC) is one of the most common malignant cancers closely associated with chronic infection by the hepatitis B virus (HBV) or the hepatitis C virus (HCV) throughout the world. Differential expression of the proteome in HBV- and HCV-associated HCC was investigated to identify any useful biomarkers indicating virus-specific hepatocarcinogenesis., Experimental Design: Twenty-one pairs of specimens (tumorous and surrounding nontumorous liver tissues) were obtained from 21 HCC patients. They were divided into three HCC types by viral markers: 7 hepatitis B surface antigen-positive (B-type HCC), 7 anti-HCV-positive (C-type HCC), and 7 hepatitis B surface antigen-negative and anti-HCV-negative. Total proteins were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and alterations in the proteome were examined., Results: Sixty proteins were identified that show significant changes in the expression level between nontumorous and tumorous tissues. Among these, 14 proteins were commonly changed in all three of the HCC types, but 46 proteins showed a tendency of viral marker specificity., Conclusions: The identified proteins were classified according to the viral factor as being involved in B-type and C-type HCC. These results suggest strongly that the expression pattern of proteome in HCC tissues is closely associated with etiologic factors. The different protein profiles between B-type and C-type HCC indicate that the pathogenetic mechanisms of hepatocarcinogenesis may be different according to the viral factor, HBV and HCV.

We present two rare cases of multifocal hepatic steatosis as a variant of fatty liver. Multifocal hepatic steatosis can cause misleading findings in the differential diagnosis when using ultrasound and computed tomography. This case report describes the atypical findings of focal fatty liver infiltrations, which were misdiagnosed as diffuse metastatic disease. The correct diagnosis was established with magnetic resonance imaging using T1-weighted gradient-echo and T2-weighted Turbo spin-echo sequences with spectral fat suppression. Multifocal hepatic steatosis was proven by biopsy.

Development of hepatocellular carcinoma (HCC) is a complex process involving multiple changes in gene expression and usually occurs in the presence of liver cirrhosis. In this research, we observed proteome alterations of three tissue types isolated from livers of HCC patients: normal, cirrhotic, and tumorous tissue. Proteome alterations were observed using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Comparing the tissue types with each other, a significant change in expression level was found in 21 proteins. Of these proteins, sarcosine dehydrogenase, liver carboxylesterase, peptidyl-prolyl isomerase A, and lamin B1 are considered novel HCC marker candidates. In particular, lamin B1 may be considered as a marker for cirrhosis, because its expression level changes considerably in cirrhotic tissue compared with normal tissue. The proteins revealed in this experiment can be used in the future for studies pertaining to hepatocarcinogenesis, or as diagnostic markers and therapeutic targets for HCC.

The aim of this study was to determine if different types of focal hepatic lesions can be differentiated by specific quantitative and qualitative imaging characteristics pre- and post-Mangafodipir trisodium (MnDPDP) administration using a computerized multivariable, discriminant analysis (DA). In a multicenter trial, 151 patients with focal liver disease were studied at 1.5 and 1.0 T using gradient-recalled echo T1 and fast spin-echo T2-weighted images pre and post MnDPDP (0.005 mmol/kg b.w.) i.v. administration. Analysis could be performed in 141 of 151 of the patients. The variables used in both single variable analysis and DA included contrast-to-noise ratios pre and post MnDPDP, presence of rim enhancement, margin, and heterogeneity of a lesion pre and post MnDPDP. The classification of diagnoses using DA was compared with a standard of reference (HCC in 23%, metastases in 25%, cyst in 13%, FNH in 10%, hemangioma in 11%, and other or no lesion in 18% of the patients; histology in 49%, long-term follow-up in 51% of the cases). In the differentiation of the various hepatic lesions, CNR together with the presence of heterogeneity or rim enhancement as variables for DA gave the highest sensitivity, specificity, and accuracy which ranged between 65 and 93, 44 and 83, and 65 and 86%, respectively. The DA models based on post-MnDPDP variables showed better classification results than the models based on pre-MnDPDP variables. An improvement of accuracy was observed when differentiating HCC from FNH lesion groups (48.9-67.4%; p < or = 0.05), and when differentiating HCC from metastasis lesion groups (68.3-84.1%; p < or = 0.01). In all regards there was no difference for T2-weighted images pre and post MnDPDP. By combining quantitative and qualitative variables, DA proved to be a useful tool in lesion discrimination. Due to considerable heterogeneity within some of the lesion type groups, the definite diagnostic impact of MnDPDP cannot be completely established yet, and further investigation is still necessary.

Rationale and Objectives: The authors evaluate the value of two fast spin-echo sequences (FSE) with different T2-weighting (repetition time [TR]/echo time [TE] = 2000/90 mseconds and TR/TE = 2000/40 mseconds) and combined gradient-and-spin-echo sequences (TR/TE = 2000/90 mseconds) for contrast-enhanced liver imaging with superparamagnetic iron oxide (AMI-25)., Methods: Forty-seven patients with focal liver lesions underwent magnetic resonance imaging at 1.0 tesla. AMI-25 was administered intravenously at a dose of 15 micromol iron/kg., Results: Administration of AMI-25 resulted in a significant increase of lesion/liver contrast-to-noise ratio (C/N) for all T2-weighted sequences (P < 0.001). On the precontrast images, the FSE sequence with a TE of 90 mseconds had the highest C/N (16.0 +/- 4.5) whereas the best postcontrast C/N (27.9 +/- 7.6) was obtained with the mild T2-weighted FSE sequence with a TE of 40 mseconds., Conclusions: Fast spin-echo sequences are valuable sequences for imaging of the liver at 1.0 tesla. For AMI-25-enhanced magnetic resonance imaging, a mild T2-weighted FSE sequence is recommended.

Purpose: Our goal was to investigate the value of T2-weighted fast SE (FSE) sequences, FSE sequences with shortened echo spacing (UFSE), combined gradient and spin echo sequences (GRASE), and segmented T1-weighted echo planar imaging (EPI) in comparison with conventional SE sequences in the detection of focal liver lesions., Method: Thirty-five patients with malignant focal liver lesions underwent MRI at 1.0 T., Results: All fast T2-weighted imaging techniques (FSE, UFSE, GRASE) showed fewer artifacts and overall better image quality than the conventional SE sequence. Quantitative analysis demonstrated that UFSE imaging had the highest tumor/liver contrast/noise ratio (C/N) (13.9 +/- 5.5) followed by FSE (12.7 +/- 4.5), T2 SE (10.9 +/- 4.2), and GRASE (10.0 +/- 4.8) sequences. The differences in C/N between the UFSE and T2 SE sequences was statistically significant (p < 0.05). C/N was significantly higher (p < 0.01) for the T1 SE (-7.4 +/- 3.8) than for the EPI sequence (-0.5 +/- 5.6)., Conclusion: FSE sequences with shortened echo spacing are valuable for liver imaging at 1.0 T. In comparison with the T2 SE sequence, they yielded better image quality and comparable tumor/liver C/N.

The purpose of our work was to provide data on the recurrence of resected benign hepatic lesions and to evaluate the value of follow-up examinations in this group of patients. From August to October 1993, 75 patients who had been admitted for liver surgery for benign tumors between 1975 and 1993 were controlled by physical examinations, serological tests, US, and, in the case of equivocal US findings, by CT. The histological diagnoses of the operative specimen included hydatidosis in 43 patients, focal nodular hyperplasia (FNH) in 12 patients, liver cell adenoma in 8 patients, cavernous hemangioma in 8 patients, and congenital cyst in 4 patients. Hepatic scars were observed in 36 of the 75 patients. Four cases of intrahepatic recurrence and 1 case of intraperitoneal spread were observed in the 42 patients with recent hydatosis. Long-term postoperative controls (specific serological tests, US) are necessary in the management of patients with hydatid disease. Follow-up examinations are not indicated in asymptomatic patients who have been operated on for FNH, hemangioma, or congenital cysts.

Purpose: The purpose of this study was to determine the ability of noninvasive imaging methods to localize focal liver lesions to specific hepatic segments., Material and Methods: In a prospective study we evaluated 24 patients with hepatic masses with ultrasonography (US), spiral CT and MR imaging., Results: The primary segmental location of the lesions was correct with US in 15 of 24 patients (63%), with CT in 21 of 24 patients (88%) and with MR imaging in 17 of 22 patients (77%). The full extent of the lesions was correctly described with US in 9 of 24 patients (38%), with CT in 16 of 24 patients (67%) and with MR in 12 of 22 patients (55%)., Conclusion: Among the noninvasive imaging methods, CT provides the best information for determining the segmental location and planning the surgical approach to hepatic resections.

Purpose: To evaluate the value of rapid T2-weighted turbo-spin sequences (TSE), turbo-spin-echo sequences with shortened echo spacing (UTSE), gradient-and-spin-echo sequences (GraSE) and T1-weighted echo-planar imaging (EPI) in comparison with conventional spin-echo sequences (SE) in the diagnosis of focal liver lesions., Methods: 20 patients with malignant focal liver lesions underwent magnetic resonance imaging at 1.0 tesla., Results: The use of fast T2-sequences (TSE, UTSE, GraSE) reduced the examination time to about 35-50%. Artifacts were reduced compared with the conventional T2-SE sequence. Quantitative analysis demonstrated that UTSE imaging had the highest tumor/liver contrast-to-noise ratio (CNR) followed by TSE, T2-SE and GraSE sequences. CNR with EPI was lower than with the T1-SE sequence (p < 0.05)., Conclusion: Turbo-spin-echo sequences with shortened echo spacing yield a shorter imaging time and improved image quality without loss of signal intensity in tumor/liver-CNR, compared with conventional T2-pulse sequences in liver imaging at 1.OT.