Circulating microRNA-200 Family as Diagnostic Marker in Hepatocellular Carcinoma

Goals

In this clinical study, we aimed to evaluate the role of circulating microRNA-200 family as a non-invasive tool to identify patients with cirrhosis-associated hepatocellular carcinoma (HCC).

Background

Prognosis of HCC remains poor with increasing incidence worldwide, mainly related to liver cirrhosis. So far, no reliable molecular targets exist for early detection of HCC at surgically manageable stages. Recently, we identified members of the microRNA-200 family as potential diagnostic markers of cirrhosis-associated HCC in patient tissue samples. Their value as circulating biomarkers for HCC remained undefined.

Methods

Blood samples and clinicopathological data of consecutive patients with liver diseases were collected prospectively. Expression of the microRNA-200 family was investigated by qRT-PCR in blood serum samples of 22 HCC patients with and without cirrhosis. Serum samples of patients with non-cancerous chronic liver cirrhosis (n = 22) and of healthy volunteers (n = 15) served as controls.

Results

MicroRNA-141 and microRNA-200a were significantly downregulated in blood serum of patients with HCC compared to liver cirrhosis (p<0.007) and healthy controls (p<0.002). MicroRNA-141 and microRNA-200a could well discriminate patients with cirrhosis-associated HCC from healthy volunteers with area under the receiver-operating characteristic curve (AUC) values of 0.85 and 0.82, respectively. Additionally, both microRNAs could differentiate between HCC and non-cancerous liver cirrhosis with a fair accuracy.

Conclusions

Circulating microRNA-200 family members are significantly deregulated in patients with HCC and liver cirrhosis. Further studies are necessary to confirm the diagnostic value of the microRNA-200 family as accurate serum marker for cirrhosis-associated HCC.     

The Singapore Liver Cancer Recurrence (SLICER) Score for Relapse Prediction in Patients with Surgically Resected Hepatocellular Carcinoma

Background and Aims

Surgery is the primary curative option in patients with hepatocellular carcinoma (HCC). Current prognostic models for HCC are developed on datasets of primarily patients with advanced cancer, and may be less relevant to resectable HCC. We developed a postoperative nomogram, the Singapore Liver Cancer Recurrence (SLICER) Score, to predict outcomes of HCC patients who have undergone surgical resection.

Methods

Records for 544 consecutive patients undergoing first-line curative surgery for HCC in one institution from 1992–2007 were reviewed, with 405 local patients selected for analysis. Freedom from relapse (FFR) was the primary outcome measure. An outcome-blinded modeling strategy including clustering, data reduction and transformation was used. We compared the performance of SLICER in estimating FFR with other HCC prognostic models using concordance-indices and likelihood analysis.

Results

A nomogram predicting FFR was developed, incorporating non-neoplastic liver cirrhosis, multifocality, preoperative alpha-fetoprotein level, Child-Pugh score, vascular invasion, tumor size, surgical margin and symptoms at presentation. Our nomogram outperformed other HCC prognostic models in predicting FFR by means of log-likelihood ratio statistics with good calibration demonstrated at 3 and 5 years post-resection and a concordance index of 0.69. Using decision curve analysis, SLICER also demonstrated superior net benefit at higher threshold probabilities.

Conclusion

The SLICER score enables well-calibrated individualized predictions of relapse following curative HCC resection, and may represent a novel tool for biomarker research and individual counseling.     

Prediction of Clinical Outcomes in Hepatitis B E Antigen Negative Chronic Hepatitis B Patients with Elevated Hepatitis B Virus DNA Levels

Objectives

We investigated whether long-term clinical outcomes such as disease progression or inactive hepatitis B virus (HBV) carrier state can be predicted by baseline factors in hepatitis B e antigen (HBeAg)-negative HBV infected patients with an elevated viral load.

Methods

A retrospective cohort of 527 HBeAg-negative chronic HBV infected patients with an elevated viral load (HBV DNA ≥ 2,000 IU/ml) was assessed for disease progression defined by the development of hepatocellular carcinoma (HCC) or cirrhotic complication, as well as becoming an inactive carrier.

Results

During a median 3.6 years of follow-up, disease progression was detected in 46 patients (40 with HCC, 6 with cirrhotic complication), and 31 of 309 non-cirrhotic patients became inactive carriers. Older age, male gender, cirrhosis, high HBV DNA levels at baseline, and short antiviral therapy duration were independent risk factors for HCC. Low HBV DNA and quantitative hepatitis B surface antigen (qHBsAg) levels were independent predictors for becoming inactive carriers in patients without cirrhosis. In non-cirrhotic patients with both low qHBsAg and HBV DNA levels, the 5-year cumulative incidence of an inactive carrier was 39.8%, while that of disease progression was 1.6%.

Conclusion

HBeAg negative patients without cirrhosis can be closely monitored for becoming an inactive carrier when both HBV DNA and qHBsAg levels are low, as the risk of disease progression is low while incidence of an inactive carrier is high.     

Gamma-Smooth Muscle Actin Expression Is Associated with Epithelial-Mesenchymal Transition and Stem-Like Properties in Hepatocellular Carcinoma

Background and Aims

The prognosis of hepatocellular carcinoma (HCC) is hampered by frequent tumour recurrence and metastases. Epithelial-Mesenchymal Transition (EMT) is now recognized as a key process in tumour invasion, metastasis and the generation of cancer initiating cells. The morphological identification of EMT in tumour samples from the expression of novel mesenchymal markers could provide relevant prognostic information and aid in understanding the metastatic process.

Methods

The expression of Smooth Muscle Actins was studied using immunofluorescence and immunohistochemistry assays in cultured liver cells during an induced EMT process and in liver specimens from adult and paediatric HCC series.

Results

We report here that in HCC cell lines treated with TGF-β and in HCC specimens, the expression of αSMA, a known mesenchymal marker of EMT, could never be detected. In addition, our in vitro studies identified the enteric form of SMA, γSMA, as being a marker of EMT. Moreover, this SMA isoform was expressed in 46% of 58 tumours from 42 adult HCC patients and in 90% of 16 tumours from 12 paediatric HCC patients. Interestingly, this expression was significantly correlated with poor tumour differentiation and progenitor cell features characterized by the expression of EpCAM and K19.

Conclusion

Taken together, our results support the conclusion that γSMA expression in HCC is strongly correlated with the EMT process, HCC aggressiveness and the identification of cancer stem cells. This correlation suggests that γSMA represents a novel and powerful marker to predict HCC progression.     

Evolutionary Changes of Hepatitis B Virus Pre-S Mutations Prior to Development of Hepatocellular Carcinoma

Background and Aims

Deletions/mutations in the hepatitis B virus (HBV) pre-S region have been associated with hepatocellular carcinoma (HCC). We aimed to study the evolutionary changes of pre-S mutations prior to HCC development.

Methods

We studied the HBV pre-S sequences at 1 to 10 years preceding diagnosis of HCC in 74 patients with HBV-related HCC (HCC group). 148 chronic hepatitis B patients matched for sex and age in 2:1 ratio, who had been followed up for at least 3 years without HCC (HCC-free group) were recruited as controls. 56 and 47 patients of HCC and HCC-free groups respectively had serially stored sera for longitudinally examination at 1–3 years, 4–6 years, 7–9 years and ≥10 years prior to the recruitment of the study.

Results

Compared to the HCC-free group, higher frequencies of pre-S deletions and point mutations (at 11 codons) were observed in the HCC group (p<0.05). Multiple logistic regression analysis showed that pre-S deletions, point mutations at codon 51 and 167 were independent factors associated with HCC. Longitudinal observation showed that pre-S deletions and most of the 11 HCC-associated pre-S point mutations existed at least 10 years before HCC development, and were more prevalent preceding HCC development in patients from HCC groups than HCC-free group. The number of HCC-associated pre-S point mutations increased over time preceding HCC development, and correlated positively with the time to HCC diagnosis (r = 0.220, p = 0.005).

Conclusions

High prevalence and cumulative evolution of pre-S mutations preceding HCC development suggested a possible carcinogenic role of pre-S mutations and their potential application in HCC risk prediction.     

Tie2-Expressing Monocytes Are Associated with Identification and Prognoses of Hepatitis B Virus Related Hepatocellular Carcinoma after Resection

Background

Tie2-expressing monocytes (TEMs) are found in various tumors, involved in forming tumor blood vessels and expressing several important proangiogenic factors. The goals of this study were to evaluate the value of TEMs in diagnosing and predicting the prognosis of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC).

Methods

Flow cytometry was performed to identify and count TEMs in peripheral blood monocytes from HCC patients (n = 84) receiving hepatectomy, HBV cirrhotic patients (n = 21), benign tumors patients (n = 15) and healthy volunteers (n = 23). Angiopoietin-2 (Ang-2) levels in the plasma were determined by enzyme linked immunosorbent assay. The distribution of TEMs in tumor tissue was observed by immunofluorescence staining. Then we determined the vascular area as a percentage of tumor area (vascular area/tumor area) by immunohistochemical staining. Finally the prognostic significance of TEMs and other clinicopathologic factors was evaluated.

Results

Percentage of TEMs in peripheral blood monocytes significantly increased in HCC patients compared with HBV cirrhotic patients and healthy donors (both P< 0.001). However there was no significance in benign liver tumor (P = 0.482). In addition, the percentage of circulating TEMs was positively correlated with plasma Ang-2 concentration (P<0.001, r² = 0.294) and vascular area/tumor area (P<0.001, r² = 0.126). Furthermore the percentage of intratumoral TEMs was significantly higher than that of paratumoral TEMs (P<0.001). Increased circulating TEMs was associated with poor overall survival (P = 0.043) and a shorter time to recurrence (P = 0.041). Multivariate Cox analysis also revealed that the percentage of TEMs in peripheral blood was an independent factor for HCC patients’ prognosis.

Conclusions

TEMs may promote angiogenesis in HCC regarding the angiopoietin/Tie2 signal pathway. Percentage of TEMs in peripheral blood monocytes may be applied as a biomarker for identifying HBV-related HCC and predicting the prognosis of these patients after resection.     

Identifying Prognostic Features by Bottom-Up Approach and Correlating to Drug Repositioning

Background

Traditionally top-down method was used to identify prognostic features in cancer research. That is to say, differentially expressed genes usually in cancer versus normal were identified to see if they possess survival prediction power. The problem is that prognostic features identified from one set of patient samples can rarely be transferred to other datasets. We apply bottom-up approach in this study: survival correlated or clinical stage correlated genes were selected first and prioritized by their network topology additionally, then a small set of features can be used as a prognostic signature.

Methods

Gene expression profiles of a cohort of 221 hepatocellular carcinoma (HCC) patients were used as a training set, ‘bottom-up’ approach was applied to discover gene-expression signatures associated with survival in both tumor and adjacent non-tumor tissues, and compared with ‘top-down’ approach. The results were validated in a second cohort of 82 patients which was used as a testing set.

Results

Two sets of gene signatures separately identified in tumor and adjacent non-tumor tissues by bottom-up approach were developed in the training cohort. These two signatures were associated with overall survival times of HCC patients and the robustness of each was validated in the testing set, and each predictive performance was better than gene expression signatures reported previously. Moreover, genes in these two prognosis signature gave some indications for drug-repositioning on HCC. Some approved drugs targeting these markers have the alternative indications on hepatocellular carcinoma.

Conclusion

Using the bottom-up approach, we have developed two prognostic gene signatures with a limited number of genes that associated with overall survival times of patients with HCC. Furthermore, prognostic markers in these two signatures have the potential to be therapeutic targets.     

Clinical Outcomes of Patients with Resected Oral Cavity Cancer and Simultaneous Second Primary Malignancies

Objectives

Simultaneous second primary tumors (SSPT) are not uncommon in patients with oral cavity squamous cell carcinoma (OSCC) living in areas where the habit of betel quid chewing is widespread. We sought to identify the main prognostic factors in OSCC patients with SSPT and incorporate them into a risk stratification scheme.

Methods

A total of 1822 consecutive patients with primary OSCC treated between January 1996 and February 2014 were analyzed for the presence of SSPT. The 18-month and 5-year overall survival (OS) rates served as the main outcome measures.

Results

Of the 1822 patients, 77 (4%) were found to have SSPT (i.e, two malignancies identified within one month of each other). The 18-month and 5-year OS rates in patients without SSPT and with SSPT were 82% and 69%, and 72% and 53%, respectively (p = 0.0063). Patients with SSPT were further divided into patients with either esophageal cancer or hepatocellular carcinoma (eso-HCC subgroup, n = 8) and other tumors (NO eso-HCC subgroup, n = 69). After multivariate analysis, neck nodal extracapsular spread (ECS, n = 18) and the presence of eso-HCC were identified as independent adverse prognostic factors. The 18-month OS rates of SSPT patients with both eso-HCC and ECS (n = 5) vs. the remaining patients (n = 72) were 0% and 78%, respectively (p < 0.0001).

Conclusion

OSCC patients with neck nodal ECS and esophageal cancer or hepatocellular carcinoma as SSPT have a dismal short-term prognosis.     

Geographic Variation of Intrahepatic Cholangiocarcinoma,Extrahepatic Cholangiocarcinoma,and Hepatocellular Carcinoma in the United States

Background

Intrahepatic (ICC) and extrahepatic cholangiocarcinomas (ECC) are tumors that arise from cholangiocytes in the bile duct, but ICCs are coded as primary liver cancers while ECCs are coded as biliary tract cancers. The etiology of these tumors is not well understood. It has been suggested that the etiology of ICC is more similar to that of another type of liver cancer, hepatocellular carcinoma (HCC), than to the etiology of ECC. If this is true, geographic incidence patterns and trends in ICC incidence should be more similar to that of HCC than ECC.

Methods

To examine this hypothesis, data from the North American Association of Central Cancer Registries Cancer in North America data file were analyzed. Incidence rates and joinpoint trends were calculated by demographic subgroup. County-level incidence rates were mapped.

Results

Overall incidence rates, racial distribution, male:female ratio, and peak ages were more similar between ICC and ECC than with HCC. During 2000–2009, average annual incidence rates of ECC increased. During 2005–2009, average annual ICC incidence rates also increased. High rates for all three cancer sites were found in the Pacific region, particularly Hawaii and Alaska. Rates of ICC and ECC were also high in the Northeast and the upper Midwest, while rates of HCC were high in the South.

Conclusions

Demographic patterns and geographical variation were more closely related between ICC and ECC than HCC, suggesting that the etiology of ICC and ECC may be similar. Increasing rates of both tumors suggest that further etiology studies are warranted.     

A Non-Synonymous Single Nucleotide Polymorphism in the HJURP Gene Associated with Susceptibility to Hepatocellular Carcinoma among Chinese

Objective

HJURP (Holliday Junction-Recognizing Protein) plays dual roles in DNA repair and in accurate chromosome segregation during mitosis. We examined whether the single nucleotide polymorphisms (SNPs) of HJURP were associated with the risk of occurrence of hepatocellular carcinoma (HCC) among chronic hepatitis B virus (HBV) carriers from well-known high-risk regions for HCC in China.

Methods

Twenty-four haplotype-tagging SNPs across HJURP were selected from HapMap data using the Haploview software. We genotyped these 24 SNPs using the using Sequenom''s iPLEX assay in the Fusui population, consisting of 348 patients with HCC and 359 cancer-free controls, and further investigated the significantly associated SNP using the TaqMan assay in the Haimen population, consisting of 100 cases and 103 controls. The genetic associations with the risk of HCC were analyzed by logistic regression.

Results

We observed an increased occurrence of HCC consistently associated with A/C or C/C genotypes of the non-synonymous SNP rs3771333 compared with the A/A genotype in both the Fusui and Haimen populations, with a pooled odds ratio 1.82 (95% confidence interval, 1.33–2.49; P = 1.9 × 10⁻⁴). Case-only analysis further indicated that carriers of the at-risk C allele were younger than those carrying the A/A genotype (P = 0.0016). In addition, the expression levels of HJURP in C allele carriers were lower than that in A/A genotype carriers (P = 0.0078 and 0.0010, for mRNA and protein levels, respectively).

Conclusion

Our findings suggest that rs3771333 in HJURP may play a role in mediating the susceptibility to HCC among Chinese.     

Evaluation of the Medicinal Herb Graptopetalum paraguayense as a Treatment for Liver Cancer

Background

Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third most common cause of cancer-related death worldwide. Sorafenib is the only drug for patients with advanced-stage hepatocellular carcinoma (HCC) that has been shown to confer a survival benefit to patients with HCC; however, it has many side effects. Thus, alternate therapeutic strategies with improved safety and therapeutic efficacy for the management of HCC should be developed.

Methods and Findings

We demonstrate that an extract of Graptopetalum paraguayense (GP) down-regulated the expression levels of several onco-proteins, including AURKA, AURKB, and FLJ10540, in HCC cells. To isolate the active components in the GP extracts, we prepared extracts fractions and assessed their effects on the expression of onco-proteins in HCC cells. The fraction designated HH-F3 was enriched in active ingredients, exhibited cytotoxic effects, and suppressed the expression of the onco-proteins in HCC cells. The structure of the main active compound in HH-F3 was found to be similar to that of the proanthocyanidin compounds derived from Rhodiola rosea. In addition, a distinct new compound rich in 3, 4, 5-trihydroxy benzylic moieties was identified in the HH-F3 preparations. Mechanistic studies indicated that HH-F3 induced apoptosis in HCC cells by promoting the loss of mitochondrial membrane potential and the production of reactive oxygen species. HH-F3 also enhanced PTEN expression and decreased AKT phosphorylation at Ser473 in a concentration-dependent manner in HCC cells. Moreover combination of GP or HH-F3 and sorafenib synergistically inhibits the proliferation of Huh7 cells. The treatment of a rat model with diethylnitrosamine (DEN)-induced liver cancer with extracts of GP and HH-F3 decreased hepatic collagen contents and inhibited tumor growth.

Conclusions

These results indicate that GP extracts and HH-F3 can protect the liver by suppressing tumor growth; consequently, these compounds could be considered for the treatment of HCC.     

A genetic variant in the promoter region of miR-106b-25 cluster and risk of HBV infection and hepatocellular carcinoma

Background

MiR-106b-25 cluster, hosted in intron 13 of MCM7, may play integral roles in diverse processes including immune response and tumorigenesis. A single nucleotide polymorphism (SNP), rs999885, is located in the promoter region of MCM7.

Methods

We performed a case-control study including 1300 HBV-positive hepatocellular carcinoma (HCC) cases, 1344 HBV persistent carriers and 1344 subjects with HBV natural clearance to test the association between rs999885 and the risk of HBV persistent infection and HCC. We also investigated the genotype-expression correlation between rs999885 and miR-106b-25 cluster in 25 pairs of HCC and adjacent non-tumor liver tissues.

Results

Compared with the HBV natural clearance subjects carrying rs999885 AA genotype, those with AG/GG genotypes had a decreased risk of chronic HBV infection with an adjusted odds ratio (OR) of 0.79 [95% confidence intervals (CIs) = 0.67–0.93]. However, the AG/GG genotypes were significantly associated with an increased HCC risk in HBV persistent carriers (adjusted OR = 1.25, 95% CIs = 1.06–1.47). Expression analysis revealed that the expression level of miR-106b-25 cluster was significantly higher in AG/GG carriers than those in AA carriers in non-tumor liver tissues.

Conclusions

These findings indicate that the A to G base change of rs999885 may provide a protective effect against chronic HBV infection but an increased risk for HCC in HBV persistent carriers by altering the expression of the miR-106b-25 cluster.     

Reactivation of Hepatitis B Virus in HBsAg-Negative Patients with Hepatocellular Carcinoma

Background & Aims

Despite increasing attention to hepatitis B virus (HBV) reactivation in hematologic settings, information on reactivation in hepatitis B surface (HBsAg)-negative patients with hepatocellular carcinoma (HCC) remains unknown. This study aimed to determine the incidence and risk factors of HBV reactivation in HBsAg-negative patients undergoing transarterial chemoembolization (TACE).

Methods

A total of 109 HBsAg-negative patients with HCC were consecutively recruited for this study and treated with either mono- (n = 75), combination-drug TACE (n = 20), or combination-drug TACE plus radiotherapy (n = 14). With serial monitoring of virological markers every 2–3 months, patients were observed for HBV reactivation (defined as the reappearance of HBV DNA or sero-reversion of HBsAg) in comparison with control subjects with HBsAg-negative cirrhosis (n = 16) or HBsAg loss (n = 46).

Results

During the study period, HBV reactivation occurred in 12 (11.0%) and 1 (1.6%) patients in the TACE and control groups, respectively. The median level of HBV DNA at reactivation was 5,174 copies/ml (range: 216–116,058). Of the 12 patients with HBV reactivation, four (33.3%) developed clinical hepatitis, including one patient who suffered from decompensation. All antiviral-treated patients achieved undetectable HBV DNA or HBsAg loss after commencement of antiviral drugs. TACE was significantly correlated with a high incidence of HBV reactivation, with increasing risk of reactivation with intensive treatment. On multivariate analysis, treatment intensity and a prior history of chronic hepatitis B remained independently predictive of reactivation.

Conclusions

TACE can reactivate HBV replication in HBsAg-negative patients, with a dose-risk relationship between treatment intensity and reactivation. Patients with prior chronic HBV infection who are to undergo intensive TACE should be closely monitored, with an alternative approach of antiviral prophylaxis against HBV reactivation.     

A genetic variant in long non-coding RNA HULC contributes to risk of HBV-related hepatocellular carcinoma in a Chinese population

Background

Recently, several studies have demonstrated that two long non-coding RNAs (lncRNAs), HULC and MALAT1, may participate in hepatocellular carcinoma (HCC) development and progression. However, genetic variations in the two lncRNAs and their associations with HCC susceptibility have not been reported. In this study, we hypothesized that single nucleotide polymorphisms (SNPs) in HULC and MALAT1 may contribute to HCC risk.

Methods

We conducted a case-control study and genotyped two SNPs, rs7763881 in HULC and rs619586 in MALAT1, in 1300 HBV positive HCC patients, 1344 HBV persistent carriers and 1344 subjects with HBV natural clearance to test the associations between the two SNPs and susceptibility to HCC and HBV chronic infection.

Results

The variant genotypes of rs7763881 were significantly associated with decreased HCC risk in a dominant genetic model [AC/CC vs. AA: adjusted odds ration (OR)  =  0.81, 95% confidence intervals (CIs)  =  0.68–0.97, P  =  0.022]. Furthermore, the variant genotypes of rs619586 was associated with decreased HCC risk with a borderline significance (AG/GG vs. AA: adjusted OR  =  0.81, 95% CIs  =  0.65–1.01, P  =  0.057). However, no significant association was found between the two SNPs and HBV clearance.

Conclusions

The variant genotypes of rs7763881 in HULC may contribute to decreased susceptibility to HCC in HBV persistent carriers.     

Diabetes Mellitus Is Associated with Hepatocellular Carcinoma: A Retrospective Case-Control Study in Hepatitis Endemic Area

Background

A number of case-control patient studies have been conducted to investigate the association between diabetes mellitus (DM) and hepatocellular carcinoma (HCC). Despite some controversial reports, it has been suggested that DM is associated with HCC. The previous studies on this subject vary in the selection of populations, sample sizes, methodology, and analysis results. Therefore, it is necessary to further delineate the involvement of DM, together with other related risk factors, in HCC with large sample size and strict analysis methodology.

Methods

We conducted a hospital-based retrospective case-control study at Perking Union Medical College Hospital, China. A total of 1,568 patients with liver diseases were enrolled in the statistical study to evaluate the association of DM and other risk factors with HCC. Among these patients, 716 of them were diagnosed with benign liver diseases, and 852 patients were diagnosed as HCC. We utilized binary logistic regression and stepwise logistic regression to investigate the associations among DM, hypertension, fatty liver, cirrhosis, gallstone, HBV infection, HCV infection, and HCC.

Results

Statistical analysis through the stepwise regression model indicated that the prevalence of DM, male gender, cirrhosis, HCV infection, or HBV infection is higher in the HCC patient group compared to the control group. However, the prevalence of gallstone is negatively associated with HCC cases. DM co-exists with HBV infection, male gender, and age in the HCC cases. Binary logistic regression analysis suggested that DM may synergize with HBV infection in HCC development.

Conclusion

DM is strongly associated with the increased risk of HCC regardless of the prevalence of HBV infection, HCV infection, cirrhosis, male gender, and age. However, the synergistic interaction between DM and HBV in HCC occurrence is significant. Therefore, DM patients with HBV infection represent a very high HCC risk population and should be considered for HCC close surveillance program.     

Distinct Distribution Pattern of Hepatitis B Virus Genotype C and D in Liver Tissue and Serum of Dual Genotype Infected Liver Cirrhosis and Hepatocellular Carcinoma Patients

Aims

The impact of co-infection of several hepatitis B virus (HBV) genotypes on the clinical outcome remains controversial. This study has for the first time investigated the distribution of HBV genotypes in the serum and in the intrahepatic tissue of liver cirrhotic (LC) and hepatocellular carcinoma (HCC) patients from India. In addition, the genotype-genotype interplay and plausible mechanism of development of HCC has also been explored.

Methods

The assessment of HBV genotypes was performed by nested PCR using either surface or HBx specific primers from both the circulating virus in the serum and replicative virus that includes covalently closed circular DNA (cccDNA) and relaxed circular DNA (rcDNA) of HBV from the intrahepatic tissue. The integrated virus within the host chromosome was genotyped by Alu-PCR method. Each PCR products were cloned and sequences of five randomly selected clones were subsequently analysed.

Results

HBV/genotype D was detected in the serum of all LC and HCC patients whereas the sequences of the replicative HBV DNA (cccDNA and rcDNA) from the intrahepatic tissue of the same patients revealed the presence of both HBV/genotype C and D. The sequences of the integrated viruses exhibited the solo presence of HBV/genotype C in the majority of LC and HCC tissues while both HBV/genotype C and D clones were found in few patients in which HBV/genotype C was predominated. Moreover, compared to HBV/genotype D, genotype C had higher propensity to generate double strand breaks, ER stress and reactive oxygen species and it had also showed higher cellular homologous-recombination efficiency that engendered more chromosomal rearrangements, which ultimately led to development of HCC.

Conclusions

Our study highlights the necessity of routine analysis of HBV genotype from the liver tissue of each chronic HBV infected patient in clinical practice to understand the disease prognosis and also to select therapeutic strategy.     

The Predictive Value of Golgi Protein 73 in Differentiating Benign from Malignant Liver Tumors

Introduction

In the work up of primary solid liver lesions it is essential to differentiate correctly between benign and malignant tumors, such as hepatocellular adenoma (HCA) and hepatocellular carcinoma (HCC) respectively. A promising new marker to detect HCC is Golgi Protein 73 (GP73). Studies comparing patients with HCC and cirrhosis with normal controls suggested that GP73 is specific for patients with HCC; however, patients with other liver tumors were not included. We therefore studied the predictive value of GP73 in differentiating between solid benign and malignant liver tumors.

Materials and Methods

This study included 264 patients: 88 patients with HCC, 88 with hepatocellular adenoma (HCA), and 88 with focal nodal hyperplasia (FNH). A blood sample was collected from each patient to measure GP73 levels using a quantitative ELISA assay and differences in outcome between subgroups were compared. The receiver operating characteristic (ROC) curve, sensitivity and specificity of GP73 were calculated and compared to alpha-fetoprotein (AFP) levels.

Results

When comparing malignant and benign liver tumors the area under ROC was 0.701 and 0.912 for GP73 and AFP respectively. Test characteristics revealed a sensitivity of 60% for GP73 and 65% for AFP; in addition the specificity was 77% for GP73 and 96% for AFP.

Conclusion

Although the literature suggests that GP73 is a valuable serum marker in patients with HCC, the serum concentration may also be increased in patients with solid benign liver tumors. Therefore, a GP73 assay is less suitable for discriminating between primary malignant and benign tumors of the liver.     

An intronic variant in the GRP78, a stress-associated gene, improves prediction for liver cirrhosis in persistent HBV carriers

Background

Our previous study indicated that a common variant (rs430397 G>A) in the intron 5 of glucose-regulated protein 78 (GRP78) gene was associated with risk and prognosis of primary hepatocellular carcinoma (HCC), including HBV- and cirrhosis-related HCC. rs430397 polymorphism may be a contributing factor or biomarker of HBV infection or HBV-related cirrhosis.

Methodology/Principal Findings

539 non-HBV-infected individuals, 205 self-limited infection and 496 persistent HBV infection were recruited between January 2001 and April 2005 from the hospitals in Southern China. Genomic DNA was genotyped for rs430397. The associations between the variation and susceptibility to liver cirrhosis (LC) in persistent HBV infection were examined. We observed that individuals carrying allele rs430397A were more likely to become HBV-related LC. When persistently infected patients were divided into four subgroups, patients with phase IV had an increased allele A and genotype AG compared with phase I and/or phase III. Decreased serum albumin and prolonged plasma prothrombin time (PT) were showed in LC patients carrying genotype AA. Furthermore, rs430397 genotype had an increased susceptibility to LC with dose-dependent manners (P-trend = 0.005), and the genotype did constitute a risk factor for the development of advanced LC (Child–Pugh classification C and B, P-trend = 0.021).

Conclusions/Significance

rs430397 polymorphism may be a contributing factor to LC in persistent HBV carriers.