High Frequency In Hepatocellular Carcinoma Research Articles

Clinical Significance of Telomerase Reverse-Transcriptase Promoter Mutations in Hepatocellular Carcinoma.

Simple SummaryActivating mutations in the promoter region of TERT (TERTp) gene are frequently observed in low- and high-grade dysplastic nodules and defined as early events in hepatocellular carcinoma development. This study shows that the nucleotide change G>A at position −124 in the TERTp region is very common in hepatocellular carcinoma. The concordance rate between droplet digital PCR (ddPCR) (63.6%) and Sanger sequencing (52.1%) detection methods is good (83.5%). HCC patients carrying the TERTp mutation had lower levels of the tumour biomarker Ca19-9 but showed reduced survival. The presence of TERTp mutations may represent a prognostic signature in liver cancer.Telomerase reactivation during hepatocarcinogenesis is recurrently caused by two point mutations occurring most frequently at the nucleotide −124 (95%) and occasionally at the nucleotide −146 (<5%) upstream of the TERT translational start site in hepatocellular carcinoma (HCC). In this study, we designed a droplet digital PCR (ddPCR) assay to detect TERT promoter (TERTp) nucleotide change G>A at position −124 and to quantify the mutant allele frequency (MAF) in 121 primary liver cancers, including 114 HCC along with 23 autologous cirrhotic tissues, five cholangiocarcinoma (CC), and two hepato-cholangiocarcinoma (HCC-CC). All cases were evaluated for tumour markers such as α-fetoprotein (AFP), carbohydrate antigen 19-9 (CA19-9), and carcinoembryonic antigen (CEA). We compared the sensitivity of ddPCR and Sanger sequencing and investigated the prognostic relevance of TERTp mutations. The TERTp G>A transition was identified in 63.6% and 52.1% of HCC samples by ddPCR and Sanger sequencing, respectively. One out of 23 (4.3%) peri-tumour tissues tested positive only by ddPCR. One out of five CC (20%) and none of the HCC-CC were found concordantly mutated by the two methods. The TERTp MAF ranged from 2% to 66%, and the large majority (85.5%) of mutated samples showed a value above 20%. A statistically significant correlation was found between TERTp mutation and tumour size (p = 0.048), while an inverse correlation was observed with CA19-9 levels (p = 0.0105). Moreover, HCC patients with TERTp −124A had reduced survival. In conclusion, the single nucleotide variation G>A at position −124 in TERTp, detected either by ddPCR or by Sanger sequencing, showed a remarkable high frequency in HCC. Such mutation is associated with lower levels of CA19-9 and reduced survival in HCC patients suggesting that the TERTp status may represent a distinct signature of liver cancer subgroups.

Dampened VEPH1 activates mTORC1 signaling by weakening the TSC1/TSC2 association in hepatocellular carcinoma

Abnormal activation of mTORC1 signaling occurs at high frequency in hepatocellular carcinoma (HCC). However, the underlying causes of this aberrant activation remain elusive. In this study, we identified ventricular zone expressed pleckstrin homology domain-containing 1 (VEPH1) as a novel tumor suppressor that acts via the mTORC1 axis. We performed quantitative reverse-transcription PCR (92 pairs), western blot (30 pairs), and immunostaining (225 cases) assays in HCC tissue samples to evaluate VEPH1 expression. We explored the functional effects of VEPH1 on tumor growth and metastasis. Molecular and biochemical strategies were used to gain insight into mechanisms underlying the tumor-suppressive function of VEPH1. VEPH1 is frequently silenced in HCC tissues, primarily resulting from let-7d upregulation. Decreased VEPH1 expression is associated with poor prognosis and aggressive tumor phenotypes in patients with HCC. VEPH1 mediates its tumor-suppressing activity through regulation of cell proliferation, migration and invasion invitro and invivo. The VEPH1 fragments 580-625aa and 447-579 aa bind directly to TSC1 (719-1,164aa) and TSC2 (1-420 aa), respectively, enhancing TSC1/TCS2 binding and promoting translocation of TSC2 to the membrane, which leads to increased TSC2 Ser1387 phosphorylation. Subsequently, Rheb is inactivated by the GTPase activity of TSC2, inhibiting mTORC1 signaling and contributing to changes in HCC carcinogenesis and metastasis. Rapamycin, the mTOR inhibitor, can inhibit the pro-tumorigenic effect of VEPH1 knockdown. Loss of VEPH1 correlates with decreased TSC2 Ser1387 phosphorylation and increased mTOR activity in HCC specimens. The loss of VEPH1 leads to aberrantly activated mTORC1 signaling in HCC; rapamycin (or rapalogs) may serve as an effective treatment option for patients with HCC and dampened VEPH1 expression. Abnormally activated mammalian target of rapamycin (mTOR) signaling is associated with poor tumor differentiation, early tumor recurrence and worse overall survival in patients with hepatocellular carcinoma. Herein, we identify low VEPH1 expression as a potential cause of abnormally activated mTOR signaling in hepatocellular carcinoma tissues. mTOR inhibitors could thus be an effective treatment option for patients with HCC and low VEPH1 expression.

Transcriptomic profiling of long non-coding RNAs in non-virus associated hepatocellular carcinoma.

Due to falling prevalence of viral hepatitis (VH), obesity, alcoholism and related liver diseases have become increasingly frequent and important as causes of hepatocellular carcinoma (HCC). However, mechanisms underlying hepatocarcinogenesis and tumor progression in VH-negative HCC remain poorly understood. Long non-coding RNAs (lncRNAs) have been implicated in pathogenesis of human diseases, including HCC. Here, by analyzing 20 clinical samples' RNA-sequencing data generated from 8 VH-negative and 2 VH-positive HCC patients, we have identified and characterized 1,514 candidate lncRNAs. For differentially expressed genes (DEGs) between tumor tissues and adjacent non-tumor tissues (P < 0.05, |FC| > 2), the upregulated genes were mainly involved in the cell proliferation, and the downregulated genes mediated the metabolic processes and responses to oxidative stress, inflammation and toxic substances. Furthermore, the lncRNA-mRNA co-expression network was constructed, by which two genetic aberrations with high frequency in HCC, SPATA46 and TMEM78, were identified. In addition, we identified 16 DEGs between tumor issues from VH-negative and VH-positive HCC patients with aim to explore gene expression differences that could be involved in the pathogenesis of HCC with varying etiology. In conclusion, we performed the comprehensive analysis of lncRNA and mRNA expression profiles, which could provide valuable insights into the underlying genetic alteration in non-virus associated HCC.

Liver transplantation for hepatocellular carcinoma: Management after the transplant.

Hepatocellular carcinoma (HCC) is an increasingly common indication for liver transplantation (LT) in the United States and in many parts of the world. In the last decade, significant work has been done to better understand how to risk stratify LT candidates for recurrence of HCC following transplant using a combination of biomarker and imaging findings. However, despite the high frequency of HCC in the LT population, guidance regarding posttransplant management is lacking. In particular, there is no current evidence to support specific post-LT surveillance strategies, leading to significant heterogeneity in practices. In addition, there are no current recommendations regarding recurrence prevention, including immunosuppression regimen or secondary prevention with adjuvant chemotherapy. Finally, guidance on treatment of disease recurrence is also lacking and there is significant controversy about the use of immunotherapy in transplant recipients due to the risk of rejection. Thus, outcomes for patients with recurrence are poor. This paper therefore provides a comprehensive review of the current literature on post-LT management of patients with HCC and identifies gaps in our current knowledge that are in urgent need of further investigation.

The risk of hepatocellular carcinoma in cirrhotic patients with hepatitis C and sustained viral response: Role of the treatment regimen

Previous studies have reported a high frequency of hepatocellular carcinoma (HCC) occurrence in patients with advanced liver disease, after receipt of interferon (IFN)-free therapy for hepatitis C virus (HCV) infection. Our objective was to verify and account for this phenomenon using data from the Scottish HCV clinical database. We identified HCC-naïve individuals with liver cirrhosis receiving a course of antiviral therapy in Scotland from 1997-2016 resulting in a sustained virologic response. Patients were followed-up from their treatment start date to the earliest of: date of death, date of HCC occurrence, or 31 January 2017. We used Cox regression to compare the risk of HCC occurrence according to treatment regimen after adjusting for relevant co-factors (including: demographic factors; baseline liver disease stage; comorbidities/health behaviours, virology, and previous treatment experience). HCC occurrence was ascertained through both the HCV clinical database and medical chart review. For our main analysis, treatment regimen was defined as IFN-free vs. IFN-containing. A total of 857 patients met the study criteria, of whom 31.7% received an IFN-free regimen. Individuals receiving IFN-free therapy were more likely to be: older; of white ethnicity, Child-Turcotte-Pugh B/C vs. Child-Turcotte-Pugh A; thrombocytopenic; non-genotype 3; and treatment experienced. HCC occurrence was observed in 46 individuals during follow-up. In univariate analysis, IFN-free therapy was associated with a significantly increased risk of HCC (HR: 2.48; p = 0.021). However, after multivariate adjustment for baseline factors, no significant risk attributable to IFN-free therapy persisted (aHR: 1.15, p = 0.744). These findings suggest that the higher incidence of HCC following sustained virologic response with IFN-free therapy relates to baseline risk factors/patient selection, and not the use of IFN-free therapy per se. We examined the risk of liver cancer in 857 patients with cirrhosis in Scotland who received hepatitis C antiviral therapy and achieved a cure. We compared the risk of first-time liver cancer in patients treated with the newest interferon-free regimens, to patients treated with interferon. After accounting for the different characteristics of these two treatment groups, we found no evidence that interferon-free therapy is associated with a higher risk of liver cancer.

MiR-155 promotes epithelial-mesenchymal transition in hepatocellular carcinoma cells through the activation of PI3K/SGK3/β-catenin signaling pathways.

Oncogenic mutations in PIK3CA, the gene encoding the catalytic subunit of phosphoinositide 3-kinase (PI3K), occur with high frequency in hepatocellular carcinoma (HCC). The protein kinase Akt is considered to be the primary effector of PI3K, but there is evidence to suggest that serum and glucocorticoid kinase 3 (SGK3) acts in an Akt-independent manner downstream of PI3K. In this report, we found that SGK3 promotes epithelial-mesenchymal transition (EMT) and reduces phosphorylation-dependent degradation of β-catenin in HCC cells. We determined that miR-155, previously shown to promote EMT, stimulates the expression of SGK3 by targeting and repressing P85α, thereby removing its inhibitory effect on PI3K-AKT signaling. These findings suggest that miR-155 promotes EMT and metastatic properties in HCC cells through activation of PI3K/SGK3/β-catenin signaling pathways.

Abstract 3958: The phenotypic and signaling consequences of a novel aberrantly spliced transcript of fibroblast growth factor receptor 3 in hepatocellular carcinoma

Abstract Aims: To identify the phenotypic and signaling consequences of a novel aberrantly spliced transcript of fibroblast growth factor receptor 3 (FGFR3) in hepatocellular carcinoma (HCC). Methods: The nested RT-PCR was employed to analyze the splicing of FGFR3 from 35 HCC cases and 11 cell lines. The bridging method was utilized to construct the sequence of FGFR3 Δ7-9 (deleting exon7, 8, and 9 from the full length of FGFR3 ORF). Based on the establishment of the FGFR3Δ7-9 lentiviral overexpress and knockdown models, the influence on bio-characters of HCC cell lines was inspected in vitro. The changes of tumorigenicity and metastases were examined quantitatively in vivo on nude mice using pathological study and Micro-PET/CT. Co-immunoprecipitation and surface plasmon resonance (SPR) binding analysis were employed to understand the ligand affinities between FGFR3Δ7-9 and FGFs. Interacting proteins and signal transduction pathway were detected by mass spectrometry and dual luciferase reporter assay. Results: FGFR3Δ7-9, lacking exons encoding the immunoglobulin-like domain III, was identified with high frequency in HCC. FGFR3Δ7-9 can apparently promote the proliferation, migration, and metastases of HCC cells both in vitro and in vivo. In contrast, knockdown of FGFR3Δ7-9 can restrict the abnormal malignancies of HCC cells. Co-immumoprecipation and SPR assay showed the binding affinity of FGFR3Δ7-9 to FGFs was significantly higher than that of wild type FGFR3 (P&amp;lt;0.01). More importantly, FGFR3Δ7-9 can be self-activated by self-dimerization and autophosphorylation even in the absence of ligand (FGFs). Consistent to the ligand-independence characteristic, FGFR3Δ7-9 demonstrated a stronger effect on inducing phosphorylation of ERK and AKT than wild type FGFR3, which may lead to abnormal down-stream PI3K/AKT/mTOR and ERK signal transductions. Furthermore, evidenced by tandem mass spectrum assay and Western blot, FGFR3Δ7-9 can up-regulate certain metastasis-related molecules: Snail and MMP-9. As a newly confirmed interaction protein to FGFR3Δ7-9, E-cadherin can be degraded via tyrosine phosphorylation, which may cause the abnormal cellular proliferation and migration consequently. Discussion/Conclusion: Dysregulation of mRNA splicing may generate an abnormal aberrant FGFR3 transcript, FGFR3Δ7-9. The joining of exon 6 to exon 10 in FGFR3Δ7-9 is in-frame, leading to the expression of a novel transmembrane form of FGFR3 containing an intact intracellular TK domain. As a ligand-independence or lowly ligand-dependent receptor, FGFR3Δ7-9 may function importantly in HCC tumorigenesis, proliferation, invasion and distant lung metastases. Altogether, our research strongly supports the idea that the dysregulation of FGFR3 expression by aberrant splicing of mRNA in a significant subset of HCC is an alternative pathway to neoplastic transformation. Note: This abstract was not presented at the meeting. Citation Format: Weihua Qiu, Weiping Yang, Xiaoqian Jing, Bingrui Wang, Xinyu Liu, Ding Ma, Helen Lin. The phenotypic and signaling consequences of a novel aberrantly spliced transcript of fibroblast growth factor receptor 3 in hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3958. doi:10.1158/1538-7445.AM2015-3958

Current concepts in pathophysiology and management of hepatocellular carcinoma.

Additional approaches to control malignancies are needed due to the emerging trends in the incidence of cancer of different organ sites. Due to the high frequency of hepatocellular carcinoma (HCC) and its poor prognosis, preventing HCC is an urgent priority. To explore the antioxidant and apoptotic pathways of grape seed extract (GSE) we induce HCC experimentally by diethylnitrosoamine (DEN) and treated the animals with low and high doses of GSE. The results indicate good therapeutic possibilities for GSE use in treatment of HCC., This was evidenced via regression of liver enzymes' function (ALT&AST), the HCC markers; α-fucosidase, α-fetoprotein and carcinoembrionic antigen (CEA) in HCC groups treated with the grape seed extract. Also, tumor necrosis factor (TNF-α) showed a significant decrease using GSE in HCC bearing animals. Regarding the apoptotic pathways of GSE, we found a significant down regulation of apoptosis enhancing nuclease (Aen), Bcl2-associated X protein (Bax), B-cell translocation gene 2(Btg2), Cyclin G1 (Ccng1) and Cyclin-dependent kinase inhibitor 1A (Cdkn1a) gene expression in HCC+GSE groups as compared to HCC bearing group. In the same trend, the necrotic/apoptotic rates were significantly higher in the HCC groups treated with GSE vs. the HCC bearing group. Finally, the 8-OHdG/2-dG generation decreased by 73.8% and 52.9% in HCC+GSE at low and high doses, respectively. Based on these encouraging observations, grape seed extract could be a promising natural remedy for attenuating hepatocellular carcinoma that has a great future in approaches directed towards control of HCC.

Influence of Rictor and Raptor Expression of mTOR Signaling on Long-Term Outcomes of Patients with Hepatocellular Carcinoma.

Aberrant signaling mediated by the mammalian target of rapamycin (mTOR) occurs at high frequency in hepatocellular carcinoma (HCC), indicating that mTOR is a candidate for targeted therapy. mTOR forms two complexes called mTORC1 (mTOR complexed with raptor) and mTORC2 (mTOR complexed with rictor). There are minor studies of the expression kinetics of mTORC1 and mTORC2 in HCC. We studied 62 patients with HCC who underwent curative resection. We used univariate and multivariate analyses to identify factors that potentially influence disease and overall survival after hepatectomy. The mRNA and protein levels of mTOR, rictor and raptor in cancer and non-cancer tissues were analyzed using quantitative RT-PCR, immunohistochemistry and Western blotting. High ratio of the levels of rictor and raptor mRNAs in tumors was identified as independent prognostic indicators for disease-free survival. Low and high levels of preoperative serum albumin and mTOR mRNA in the tumor, respectively, were identified as independent indicators of overall survival. HCC is likely to recur early after hepatic resection in patients with high levels of mTOR and rictor mRNAs and high rictor/raptor ratios in cancer tissues. We conclude that analysis of mTOR expression in cancer tissues represents an essential strategy to predict HCC recurrence after curative treatment.

Retinoblastoma protein potentiates the innate immune response in hepatocytes: significance for hepatocellular carcinoma.

Cancers mediated by viral etiology must exhibit deregulated cellular proliferation and evade immune recognition. The role of the retinoblastoma tumor suppressor (RB) pathway, which is lost at relatively high frequency in hepatocellular carcinoma (HCC), has recently been expanded to include the regulation of innate immune responsiveness. In this study we investigated the coordinate impact of RB-loss on cell cycle control and immune function in the liver. We found that RB depletion in hepatoma cells resulted in a compromised immunological response to multiple stimuli and reduced the potential of these cells to recruit myeloid cells. Viral-mediated liver-specific RB deletion in vivo led to the induction of genes associated with proliferation and cell cycle entry as well as the significant attenuation of genes associated with immune function, as evidenced by decreases in cytokine and chemokine expression, leukocyte recruitment, and hepatic inflammation. To determine if these changes in gene expression were instructive in human disease, we compared our liver-specific RB-loss gene signature to existing profiles of HCC and found that this signature was associated with disease progression and confers a worse prognosis. Our data confirm that RB participates in the regulation of innate immunity in liver parenchymal cells both in vitro and in vivo and to our knowledge describes the first gene signature associated with HCC that includes both immunoregulatory and proliferative genes and that can also be attributed to the alteration of a single gene in vitro.

RASSF1A Ala133Ser polymorphism is associated with increased susceptibility to hepatocellular carcinoma in a Turkish population

AimThe tumor suppressor gene Ras association domain family 1 isoform A (RASSF1A) regulates cell cycle regulation, apoptosis and microtubule stability and is inactivated by promoter hypermethylation at a high frequency in hepatocellular carcinoma (HCC). A guanine (G)/thymine (T) common single nucleotide polymorphism (SNP) at first position of codon 133 in RASSF1A gene determines an alanine (Ala) to serine (Ser) (Ala133Ser) amino acidic substitution which may alter cancer risk by influencing the function of RASSF1A protein. MethodsTo determine the association of the RASSF1A Ala133Ser polymorphism with the risk of HCC development in a Turkish population, a hospital-based case–control study was designed consisting of 236 subjects with HCC and 236 cancer-free control subjects matched for age, gender, smoking and alcohol status. The genotype frequency of the RASSF1A Ala133Ser polymorphism was determined by using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. ResultsAllele and genotype associations of RASSF1A Ala133Ser polymorphism with HCC susceptibility were observed in comparisons between the patient and control samples (P<0.001). Risk of HCC development in this Turkish population was significantly increased in carriers of the Ser133 variant allele of Ala133Ser polymorphism (Ala/Ser and Ser/Ser genotypes) when compared with homozygote Ala/Ala genotype (OR=5.47, 95% CI=3.63–8.25, P=0.001). ConclusionBecause our results suggest for the first time that the Ser133 allele of RASSF1A Ala133Ser polymorphism may be a genetic susceptibility factor for HCC in the Turkish population, further independent studies are required to validate our findings in a larger series, as well as in patients of different ethnic origins.

Proliferative Suppression by CDK4/6 Inhibition: Complex Function of the Retinoblastoma Pathway in Liver Tissue and Hepatoma Cells

Hepatocellular carcinoma is the third leading cause of cancer mortality worldwide; current chemotherapeutic interventions for this disease are largely ineffective. The retinoblastoma tumor suppressor (RB) is functionally inactivated at relatively high frequency in hepatocellular carcinoma and hepatoma cell lines. Here, we analyzed the ability of CDK4/6 inhibition to inhibit hepatocyte proliferation and the effect of RB status on this process. Hepatoma cell lines and xenograft models harboring RB knockdown and mice harboring liver-specific Rb deletion were used to define the role of RB function in response to CDK4/6 inhibition. Our study shows that CDK4/6-dependent cell cycle progression in hepatoma cells was readily arrested by inhibition of CDK4/6 by PD-0332991 or p16ink4a irrespective of RB status. Interestingly, upon CDK4/6 inhibition, p107 protein stability was dramatically increased as a function of RB loss. This engagement of compensatory mechanisms was critical for cell cycle inhibition in the absence of RB, because both the E1A oncoprotein and overexpression of E2F proteins were capable of overcoming the effect of CDK4/6 inhibition. These findings were recapitulated in xenograft models. Furthermore, to determine how these findings relate to hepatocyte proliferation in vivo, mice were exposed to carbon tetrachloride to induce liver regeneration followed by treatment with PD-0332991. This treatment significantly inhibited hepatocyte proliferation. Strikingly, this facet of PD-0332991 function was retained even in RB-deficient livers. These data show that CDK4/6 inhibition is a potent mediator of cytostasis and that RB loss can be readily compensated for in the context of both hepatoma cell lines and liver tissue.

Unique Impact of RB Loss on Hepatic Proliferation: TUMORIGENIC STRESSES UNCOVER DISTINCT PATHWAYS OF CELL CYCLE CONTROL

The retinoblastoma (RB) tumor suppressor pathway is disrupted at high frequency in hepatocellular carcinoma. However, the mechanisms through which RB modulates physiological responses in the liver remain poorly defined. Despite the well established role of RB in cell cycle control, the deletion of RB had no impact on the kinetics of cell cycle entry or the restoration of quiescence during the course of liver regeneration. Although these findings indicated compensatory effects from the RB-related proteins p107 and p130, even the dual deletion of RB with p107 or p130 failed to deregulate hepatic proliferation. Furthermore, although these findings suggested a modest role for the RB-pathway in the context of proliferative control, RB loss had striking effects on response to the genotoxic hepatocarcinogen diethylnitrosamine. With diethylnitrosamine, RB deletion resulted in inappropriate cell cycle entry that facilitated secondary genetic damage and further uncoupling of DNA replication with mitotic entry. Analysis of the mechanism underlying the differential impact of RB status on liver biology revealed that, while liver regeneration is associated with the conventional induction of cyclin D1 expression, the RB-dependent cell cycle entry, occurring with diethylnitrosamine treatment, was independent of cyclin D1 levels and associated with the specific induction of E2F1. Combined, these studies demonstrate that RB loss has disparate effects on the response to unique tumorigenic stresses, which is reflective of distinct mechanisms of cell cycle entry.

Model for End-Stage Liver Disease: Impact of the New Deceased Donor Liver Allocation Policy in São Paulo, Brazil

Since 1997, organ transplantation in Brazil has been regulated by a federal law, which was created to guarantee equal access to treatment on a national scale. Centralized deceased donor organ procurement and sharing are controlled by the Health Department of each state of the nation, following a regional allocation policy. In São Paulo, time on the waiting list was the main criterion adopted to allocate deceased donor livers up to July 15, 2006. After that, model for end-stage liver disease/pediatric end-stage liver disease (MELD/PELD) scores were the main criteria. The aim of this study was to investigate the impact of the new criteria on patient survival rates using 895 consecutive liver recipients. The 1-year patient survival rates were compared between recipients transplanted based on the waiting time policy and based on MELD/PELD scores showing similar results (69.79% vs 66.69%; P = NS). Regarding liver allocation based on MELD/PELD scores, worse survival outcomes were observed among recipients transplanted with higher MELD scores. Also, under the new criteria, a high frequency of hepatocellular carcinoma and pediatric recipients underwent transplantation.

Polymorphism attribution of cSNPs in cancer-related genes located in loss regions with a high frequency of HCC between HBV and health groups

Cancer-related genes harbored in the loss regions containing a high frequency of hepatocellular carcinoma (HCC) were selected. Related information was gathered and the coding single nucleotide polymorphism (cSNP) sequences were obtained from the single nucleotide polymorphism (SNP) database. The appropriate primers and oligonucleotide probes were then designed in accordance with the SNP sites, and subsequently, the gene chips for detecting SNPs were constructed. Genomic DNA was extracted from blood samples of healthy controls and from patients with HBV infection. The sequences, including the SNPs, were amplified via polymerase chain reaction (PCR) and labeled using digoxigenin deoxyuridine tri-phosphate (Dig-dUTP). The labeled products were then hybridized with the SNP chips. Results confirmed that the differences in allele frequencies of three SNPs EGFL3 (rs947345), Caspase9 (rs2308950), and E2F2 (rs3218171) were distinct between HBV-infected patients and controls, suggesting that these SNPs ocuring in high frequency in HBV-infected individuals may be associated with susceptibility to HCC.

High frequency of hepatocellular carcinoma in Mongolia; association with mono-, or Co-infection with hepatitis C, B, and delta viruses

To investigate the association between viral infection pattern and hepatocellular carcinoma (HCC), 292 chronic hepatitis patients, including 108 with developed HCC were screened using serological and molecular genetics methods. Viral etiology was established in 267 (91.4%), anti-HCV detected in 198 (67.8%), and HBsAg in 124 (42.5%) including 93 (74.4%) cases with HDV co-infection. HCV mono-infection predominated in both, "non-HCC" and "HCC" groups (54% and 39%, respectively) with higher frequency in the first group (P = 0.011), whereas HBV in co-infection with HDV was more frequent in HCC group (14% vs 25%, P = 0.017). Patients with HCV mono-infection were older than those with co-infection (P<0.02), had higher frequency of HCV-viraemia (82% vs 7%, P < 0.0001), and yet had significantly lower prevalence of HCC (29.6% vs. 49.1%, P = 0.003). Alpha-fetoprotein (AFP) and protein induced by vitamin K antagonist-II (PIVKA-II) were specifically elevated in 71% of HCC patients. In conclusion, although HCV monoinfection pattern predominates in Mongolia, co-infection with HBV and HDV had stronger association with HCC development at younger age. Liver tumor markers; AFP and PIVKA-II are useful tools for complex HCC-screening and clinical follow-up for chronic hepatitis patients in Mongolia.

High frequency of promoter hypermethylation of RASSF1A in tumor and plasma of patients with hepatocellular carcinoma

To determine the presence of ras association domain family 1A (RASSF1A) promoter methylation in the tumor tissues and plasma of patients with hepatocellular carcinoma (HCC). Methylation-specific polymerase chain reaction was used to detect RASSF1A methylation in DNA extracted from HCC tumors and paired plasma samples of 40 patients. The association of RASSF1A hypermethylation in tumor and plasma DNA of HCC patients with clinicopathological characteristics was also analyzed. RASSF1A promoter hypermethylation was detected in 37 of the 40 HCC tissues (92.5%). Of the paired plasma from the 40 HCC patients, aberrant methylation was detected in 17 (42.5%). No RASSF1A methylation was detected in the plasma in the absence of methylation in the corresponding tumor. The presence of RASSF1A promoter hypermethylation in plasma DNA was found to associate with HCC size of > or =4 cm (P = 0.035). RASSF1A promoter hypermethylation occurred at a high frequency in HCC. The aberrant methylation was also detectable in over 40% of matched plasma. The latter should be evaluated as a screening tool and/or prognosticator of HCC patients.

Preparation and analysis of cSNP chip on hepatocellular carcinoma-related genes

Objective To prepare a gene chip for detecting polymorphisms on coding region of genes in hepatocellular carcinoma tissuses. Methods Genes harboring in loss regions with high frequency of hepatocellular carcinoma (HCC) were selected, the related information was inquired and the cSNP sequences were obtained in the SNP database of National Center for Biotechnology Information(NCBI).Then the appropriate primers and oligonucleotide probes were designed according to the SNP sites and the gene chip for the detection of SNPs were constructed. This chip includes 48 cSNPs of 25 hepatocellular carcinoma-related genes. The PCR products labeled by Dig-dUTP were hybridized with cSNP chip. Results The sensitivity, influence of probes and reiteration of the chip were detected. The results indicated that the chip was sensitive about 6×10~(-3) ng/μl. The signal of hybridization was down with lower concentrition of probes. By the chip, 7 of polymorphisms of caspase9 ( rs2308941) C→T and DOK2(rs2242241)T→G, 6 of polymorphisms of EGFL3(rs947345)A →G,caspase9 ( rs2308938)C→G and PHGDH(rs1801955)T→A, 5 of polymorphisms of E2F2(rs3218170) G→A,4 of polymorphisms of MUTYH(rs1140507)T→C and BNIP3L(rs1055806)G→T, 1 of polymorphism of TNFRSF1B (rs1061622)T→G were detected in the tissues of 10 HCC. Samples of caspase9 ( rs2308941G) and ( rs2308941A) were verified by PCR-SSCP and sequencing. Conclusion We prepare the cSNP chip of hepatocellular carcinoma-related genes, which can accelerate the discovery of polymorphic markers on hepatocellular carcinoma.

Frequent impairment of the spindle assembly checkpoint in hepatocellular carcinoma

Chromosomal instability (CI) leading to aneuploidy is one form of genetic instability, a characteristic feature of various types of cancers. Recent work has suggested that CI can be induced by a spindle assembly checkpoint defect. The aim of the current study was to determine the frequency of a defect of the checkpoint in hepatocellular carcinoma (HCC) and to establish whether alterations of genes encoding the checkpoint were associated with CI in HCC. Aneuploidy and the function of the spindle assembly checkpoint were examined using DNA flow cytometry and morphologic analysis with microtubule disrupting drugs. To explore the molecular basis, the authors examined the expression and alterations of the mitotic checkpoint gene, BUB1, using Northern hybridization and direct sequencing in 8 HCC cell lines and 50 HCC specimens. Furthermore, the authors examined the alterations of other mitotic checkpoint genes, BUBR1, BUB3, MAD2B, and CDC20, using direct sequencing in HCC cell lines with aneuploidy. An impaired spindle assembly checkpoint was found in five (62.5%) of the eight aneuploid cell lines. Transcriptional expressions of the BUB1 gene appeared in all cell lines. While some polymorphic base changes were noted in BUB1, BUBR1, and CDC20, no mutations responsible for impairment of the mitotic checkpoint were found in either the HCC cell lines or HCC specimens, which suggests that these genes did not seem to be involved in tumor development in HCC. The loss of spindle assembly checkpoint occurred with a high frequency in HCC with CI. However, other mechanisms might also contribute to CI in HCC.

Hepatocarcinogenesis in hepatitis viral infection: lessons from transgenic mouse studies.

Over the past decade, genetically engineered mouse models have been used for studies of the mechanisms underlying human diseases. One advantage of these models is that the targeted protein executes its function in normal cells in their natural tissue microenvironments. Transgenic mouse models for human viral hepatitis have also been established and have provided new insights into the pathogenesis of hepatitis and hepatocellular carcinoma (HCC). In the search for the mechanism of hepatocarcinogenesis in hepatitis viral infection, two viral proteins, the core protein of hepatitis C virus (HCV) and the HBx protein of hepatitis B virus (HBV), have been shown to possess oncogenic potential through transgenic mouse studies, indicating the direct involvement of the hepatitis viruses in hepatocarcinogenesis. The presence of the hepatitis C virus core or HBx protein, which has an oncogenic potential, may allow some of the steps in multistep hepatocarcinogenesis to be skipped. This may explain the very high frequency of HCC in patients with HCV or HBV infection.