Identification of novel tumor markers in hepatitis C virus-associated hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common primary cancer associated frequently with hepatitis C virus (HCV). To gain insight into the molecular mechanisms of hepatocarcinogenesis, and to identify potential HCC markers, we performed cDNA microarray analysis on surgical liver samples from 20 HCV-infected patients. RNA from individual tumors was compared with RNA isolated from adjacent nontumor tissue that was cirrhotic in all of the cases. Gene expression changes related to cirrhosis were filtered out using experiments in which pooled RNA from HCV-infected cirrhotic liver without tumors was compared with pooled RNA from normal liver. Expression of approximately 13,600 genes was analyzed using the advanced analysis tools of the Rosetta Resolver System. This analysis revealed a set of 50 potential HCC marker genes, which were up-regulated in the majority of the tumors analyzed, much more widely than common clinical markers such as cell proliferation-related genes. This HCC marker set contained several cancer-related genes, including serine/threonine kinase 15 (STK15), which has been implicated in chromosome segregation abnormalities but which has not been linked previously with liver cancer. In addition, a set of genes encoding secreted or plasma proteins was identified, including plasma glutamate carboxypeptidase (PGCP) and two secreted phospholipases A2 (PLA2G13 and PLA2G7). These genes may provide potential HCC serological markers because of their strong up-regulation in more than half of the tumors analyzed. Thus, high throughput methods coupled with high-order statistical analyses may result in the development of new diagnostic tools for liver malignancies.     

Epigenetic mechanisms in hepatitis B virus-associated hepatocellular carcinoma

Chronic infection of the liver by the hepatitis B virus (HBV) is associated with increased risk for developing hepatocellular carcinoma (HCC). A multitude of st...     

Comparison of proteome between hepatitis B virus- and hepatitis C virus-associated hepatocellular carcinoma.

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.     

Preneoplastic markers of hepatitis B virus-associated hepatocellular carcinoma

Hepatitis B virus (HBV) carriers are at high risk for the development of hepatocellular carcinoma (HCC), but there are no reliable markers that will identify such high-risk carriers. The objective of this work is to identify serologic markers that may indicate the early presence of HCC. Since HBV-encoded X antigen (HBxAg) likely contributes to HCC by up- or down-regulation of host gene expression, X positive and negative HepG2 cells were made and subjected to cDNA subtraction. When specific ELISAs were constructed measuring differentially expressed antigens and corresponding antibodies, antibodies to several differentially expressed genes were detected. In cross-sectional and longitudinal studies, antibodies were predominantly present in patients with HBV-associated cirrhosis and HCC, but not in most carriers with hepatic inflammation alone or without active liver disease. Antibodies were also present in patients with hepatitis C virus (HCV)-related HCC, but rarely detected in sera from uninfected individuals, those with tumors other than HCC, or those with drug-induced hepatitis. Statistical analysis showed that HCC patients with four or more antibodies detectable before the appearance of HCC had decreased survival, suggesting that these markers may reflect stepwise hepatocarcinogenesis. Hence, these antibodies may serve as preneoplastic markers for HCC in HBV carriers with chronic liver disease, and may be identified by a simple blood test.     

Decreased expression of cytochrome P450 2E1 is associated with poor prognosis of hepatocellular carcinoma

Cytochrome P450-2E1 (CYP2E1) is one of the major hepatic enzymes involved in the metabolism of procarcinogen. Our study aimed to investigate the differential expression level of CYP2E1 and its clinicopathological significance in hepatocellular carcinoma (HCC). CYP2E1 revealed low level of expression in 70% of the tumor tissues, when compared to the adjacent nontumor tissues, at both mRNA and protein levels. The low expression of CYP2E1 was significantly correlated with the aggressive tumor phenotype, including poor differentiation status (by the Edmondson grading system) (p=0.038), absence of tumor capsule (p=0.030) and younger age of the patients (p=0.002). Multivariate analysis indicated that CYP2E1 expression level and pTNM stage were independent prognostic factors for disease-free survival. CYP2E1 was also shown to have a differential expression level in different liver tissues. The level of CYP2E1 was significantly higher in nontumor tissues from HCC patients compared to the intermediate level in cirrhosis livers from noncancer patients and normal livers from healthy persons. Tumor tissues were shown to have the lowest expression level. In conclusion, our results have shown that CYP2E1 is upregulated in the nontumor tissue and downregulated in tumor tissue, which is associated with aggressive tumor type and poor prognosis of the patients. It suggested that the differential expression of CYP2E1 may play an important role in HCC tumorigenesis.     

Aging of patients with hepatitis C virus-associated hepatocellular carcinoma: long-term trends in Japan

The incidence of hepatocellular carcinoma (HCC) in Japan has been increasing. The aim of the present study was to analyze epidemiological changes in Japanese HCC patients. A total of 463 patients with HCC diagnosed at our hospital between 1982 and 2001 were recruited for this study. Cohorts of patients with HCC were categorized into intervals of five years. The number of HBV- and HCV-associated HCC cases had decreased and increased in 1987-1991, respectively, and thereafter reached a plateau. The mean age of patients at diagnosis of HCV-associated HCC showed a steady significant increase from 60 to 68 years of age during the period, suggesting that these findings were associated with a shift toward an older-age group that had the highest rate of HCV infection. The mean age of patients with other types of HCC did not significantly change during the period. Since it is known that the prevalence of HCV infection in young Japanese persons is low and that the incidence of HCV infection is very low at present, our findings may indicate that the prevalence of HCC will decline in Japan, an advanced country with regard to HCV-associated HCC, in the near future.     

Secular trends and geographic variations of hepatitis B virus and hepatitis C virus-associated hepatocellular carcinoma in Taiwan

Etiological variations in hepatocellular carcinoma (HCC) exist across different geographic areas. To gain better control of HCC, we retrospectively studied the secular trends and geographic variations in hepatitis B virus (HBV)-related and hepatitis C virus (HCV)-related HCCs in Taiwan. A total of 18,423 HCC cases enrolled in 8 medical centers from 1981 to 2001 were reviewed. Overall, 67% of male HCC in Taiwan was related to HBV infection whereas 55.2% of female HCC in Taiwan was related to HCV infection. The mean age of patients with HBV-related HCC was 53.2 +/- 13.6 years, while the mean age of patients with HCV-related HCC was 65.1 +/- 9.1 years (p < 0.001). The male/female ratio was 6.4 for HBV-related HCC, while it was 1.7 for the HCV-related HCC (p < 0.001). The percentage of HBV-related HCC progressively decreased from 81.5 to 66.2% in males, and from 66.7 to 41.4% in females over the study period. Our study demonstrates that the percentage of HBV-related HCC has progressively decreased over the last 20 years. The relative decrease in HBV-related HCC was not due to a decrease in HBV-related HCC death. Instead, it was caused by an increase in HCV-related HCC. Prevention of new HCV infection and the treatment of chronic hepatitis C should be the primary goals, which will result in better control of HCC in the future, even in an HBV-endemic area like Taiwan.     

细胞色素P450 2E1基因多态与肝癌遗传易感性研究

目的：探讨细胞色素Ｐ４５０ ２Ｅ１基因多态与肝癌的关系。方法：应用ＰＣＲ－ＲＦＬＰ方法对８４例肝癌患者和１４４例健康对照的细胞色素Ｐ４５０ ２Ｅ１基因ＲｓａⅠ多态进行检测。结果：病例组基因型Ａ频率为７１．４３％,等位基因ｃ１频率为８４．５２％,对照组则分别为５５．５６％和７５．３５％,两组差别均有统计学意义（Ｐ〈０．０５）。其比值比分别为２．００和１．７９,提示具有基因型Ａ或等位基因ｃ１的个体患肝     

Difference in histological activity of non-tumorous liver tissue between hepatitis B virus- and hepatitis C virus-associated hepatocellular carcinoma without cirrhosis

To elucidate the difference in the liver carcinogenetic process during hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, non-tumorous liver tissues obtained from 10 patients who developed HBV-associated hepatocellular carcinoma (HCC) without cirrhosis were compared with those obtained from 26 patients who developed HCV-associated HCC without cirrhosis. The extent of fibrosis was similar in both groups. In contrast, necroinflammatory activities were significantly higher in patients with HCV than in patients with HBV. These results indicate that ongoing liver inflammation mediates the hepatocarcinogenesis more pronouncedly in HCV infection than in HBV infection.     

丙肝相关肝细胞癌患者肝动脉化疗栓塞术对丙肝病毒再激活、肝炎加重的影响

目的:探讨丙肝病毒相关肝细胞癌患者接受肝动脉化疗栓塞术（transcatheter arterial chemoembolization,TACE）对丙肝病毒再激活、肝炎加重的影响。方法:收集2013年1月1日至2017年10月31日期间,确诊为丙肝相关肝细胞癌患者的临床资料进行统计学分析。结果:纳入156例丙肝相关肝细胞癌患者资料,针对TACE术对丙肝病毒再激活风险的统计学研究显示,74例进行TACE患者有19例发生丙肝病毒再激活,82例未行TACE患者中发生丙肝病毒再激活6例,两者具有统计学差异（P=0.00）。针对TACE术后丙肝病毒再激活风险评估显示,HCV再激活组患者术前WBC计数较未激活组患者低,两者有统计学差异(P=0.02)。Lg（HCV RNA）在两组中有差异,且Lg（HCV RNA）≥4的患者发生丙肝病毒再激活的概率大于Lg（HCV RNA）＜4的患者,两者差异有统计学意义(Fisher确切检验 P=0.03)。肿瘤直径在两组中有差异,且肿瘤直径≥6 cm患者发生病毒再激活的概率大于肿瘤直径<6 cm的患者,两者差异有统计学意义(P=0.03)。Logstic多因素回归分析发现:治疗前WBC水平、Lg（HCV RNA）及肿瘤直径是丙肝病毒再激活的独立预测因素。最后,肝炎加重发生率与丙肝病毒再激活的关联性研究显示,HCV再激活组肝炎加重发生概率明显高于HCV未激活患者（P=0.01）。结论:丙肝相关肝细胞癌患者接受TACE术增加病毒再激活风险,且低WBC水平、Lg(HCV RNA)≥4、肿瘤直径≥6 cm是发生HCV病毒再激活的独立危险因素,并且可能通过HCV再激活增加肝炎加重的风险。     

Ursodiol use is possibly associated with lower incidence of hepatocellular carcinoma in hepatitis C virus-associated liver cirrhosis.

In a previous study of patients with hepatitis C virus (HCV)-associated liver cirrhosis (HCV-LC), we showed that increased liver inflammation, as assessed by higher serum alanine aminotransferase (ALT), was associated with increased risk for the development of hepatocellular carcinoma (HCC). This suggested that suppression of inflammation might inhibit HCC development in HCV-LC. Several agents have been suggested to possess chemopreventive potential against the development of HCC in chronic HCV-associated liver disease, including herbal medicines, such as Stronger-Neo-Minophagen C (glycyrrhizin) and Sho-saiko-to (TJ-9). Ursodiol [ursodeoxycholic acid (UDCA)], a bile acid widely used to treat cholestatic liver diseases, also possesses anti-inflammatory properties in liver disease. We hypothesized that suppression of liver inflammation, as assessed by decreases in serum ALT, might inhibit HCC occurrence in patients with HCV-LC. In this study, the preventive effect of UDCA on HCC was examined in patients with early-stage HCV-LC. One hundred two patients with HCV-LC (Child stage A) were treated with anti-inflammatory drugs, Stronger-Neo-Minophagen C,Sho-saiko-to, or UDCA, with the goal of lowering the average serum ALT level to <80 IU. Iftheaverage ALT level did not remain <80 IU after treatment with one agent, multiagent therapy was initiated. The patients were followed up for >5 years and were retrospectively subdivided into two groups: 56 UDCA users (group A) and 46 UDCA nonusers (group B). The mean +/- SD dosage of UDCA administered in group A was 473.7 +/- 183.0 mg/d. The average duration of UDCA administration in group A was 37.3 +/- 15.9 months over the 5-year study period. The cumulative incidence of HCC was recorded. The 5-year incidence of HCC in group A was 17.9% (10 of 56) and was significantly lower than that in group B (39.1%, 18 of 46; P = 0.025). The risk for HCC incidence, calculated by a logistic regression model, showed that the administration of UDCA significantly decreased hepatocarcinogenesis (P = 0.036). The herbal medicines used were comparable in dosage and treatment duration in the UDCA and non-UDCA groups. In conclusion, UDCA might prevent HCC development in HCV-LC. Interestingly, because the serum ALT trends over time were nearly the same in both groups, the chemopreventive effectiveness of UDCA was not accompanied by greater reductions in ALT compared with the UDCA nonusers.     

Parallel epigenetic and genetic changes in the pathogenesis of hepatitis virus-associated hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most frequent tumor types in the world, with short survival times and few treatment options. Hepatitis B virus (HBV) and hepatitis C virus (HCV) are major etiologic agents of HCC, although the associated mechanisms are incompletely understood. The available evidence suggests that both viruses promote tumorigenesis by up-regulating genes that promote hepatocellular growth and survival, and by down-regulating other genes that act as tumor suppressors and negative growth regulatory molecules. Significantly, a number of the pathways that are altered by these viruses are the same ones that accumulate genetic alterations during tumor progression. This suggests that the pathways that promote virus persistence and replication may also promote cell growth and survival. From the perspective of the virus, this promotes chronic infection, while from the perspective of the host, this promotes tumorigenesis.     

Sex-related differences in DNA copy number alterations in hepatitis B virus-associated hepatocellular carcinoma

Background: Males have a higher prevalence of hepatocellular carcinoma (HCC) than females in general,but the reasons for the sex disparity are still obscure. DNA copy number alteration (CNA) is a major featureof solid tumors including HCC, but whether CNA plays a role in sex-related differences in HCC developmenthas never been evaluated. Methods: High-resolution array comparative genomic hybridization (CGH) was usedto examine 17 female and 46 male HCC patients with chronic hepatitis B virus (HBV) infection in Shanghai,China. Two-tailed Fisher’s exact or χ2 tests was used to compare CNAs between females and males. Results:The overall frequencies and patterns of CNAs in female and male cases were similar. However, female HCCtumors presented more copy number gains compared to those in males on 1q21.3-q22 (76.5% vs. 37.0%, P =0.009), 11q11 (35.3% vs. 0.0%, P = 0.0002) and 19q13.31-q13.32 (23.5% vs. 0.0%, P = 0.004), and loss on 16p11.2(35.3% vs. 6.5%, P = 0.009). Relative to females, male cases had greater copy number loss on 11q11 (63.0% vs.17.6%, P = 0.002). Further analyses showed that 11q11 gain correlated with 19q13.31-q13.32 gain (P = 0.042),11q11 loss (P = 0.011) and 16p11.2 loss (P = 0.033), while 1q21.3-q22 gain correlated with 19q13.31-q13.32 gain(P = 0.046). Conclusions: These findings suggest that CNAs may play a role in sex-related differences in HBVassociatedHCC development.     

Expression profile of nine novel genes differentially expressed in hepatitis B virus-associated hepatocellular carcinomas

Chronic hepatitis B virus (HBV) infection is known to be one of the major causes in the development of hepatocellular carcinoma (HCC), although the biomolecular mechanism(s) involved remain unclear. To identify the cellular gene(s) involved in HBV-associated hepatocarcinogenesis, we used the mRNA differential display method and examined three paired tumor and nontumor tissues, all of which had chromosomally integrated HBV-DNA through chronic infection. Using 240 different combinations of three one-base anchored oligo-dT primers and 80 arbitrary 13-mers, genes decreased or increased in expression more than twofold between each tumor tissue and its paired nontumor tissue were identified. Twenty-nine known genes and four novel genes were differentially over-expressed in the HCC tumor tissues. In contrast, 27 known genes and five novel genes were under-expressed in those tumor tissues. The nucleotide sequences of the nine novel gene fragments were determined and their expression patterns were examined in 40 HCC samples. HA61T2, PT18, HG63T1, and HG57T1 were preferentially over-expressed in 32 cases (80%, P<0.001), 24 cases (60%), 23 cases (57.5%) and 22 cases (55%) of the 40 tumor tissues, respectively. There was an increased frequency of HG57T1 over-expression in HCC patients with HBV-positive serology and low serum alpha-feto protein (AFP) levels (P<0.05). DNT10, PT8, PT19, ENT25 and HA6T4 were under-expressed in 26 cases (65%), 23 cases (57.5%), 21 cases (53%), 20 cases (50%) and 18 cases (45%) of the 40 tumor samples, respectively. There was a strong correlation of DNT10 under-expression with high serum AFP level in HCC patients, irrespective of HBV serology (P<0.01). HA6T4 was preferentially under-expressed in HCC tumors in patients with HBV-positive serology and high serum AFP levels (P<0.05). Thus, the functional analyses of the known and novel genes identified in this study should prove valuable to further understand the mechanism(s) of hepatocarcinogenesis.     

Identification of genes preferentially methylated in hepatitis C virus‐related hepatocellular carcinoma

Chronic infections by hepatitis B virus (HBV) and hepatitis C virus (HCV) appear to be the most significant causes of hepatocellular carcinoma (HCC). Aberrant promoter methylation is known to be deeply involved in cancer, including in HCC. In this study, we analyzed aberrant promoter methylation by methylated DNA immunoprecipitation‐on‐chip analysis on a genome‐wide scale in six HCCs including three HBV‐related and three HCV‐related HCCs, six matched noncancerous liver tissues, and three normal liver tissues. Candidate genes with promoter methylation were detected more frequently in HCV‐related HCC. Candidate genes methylated preferentially to HBV‐related or HCV‐related HCCs were detected and selected, and methylation levels of the selected genes were validated by quantitative methylation analysis using MALDI‐TOF mass spectrometry using 125 liver tissue samples, including 61 HCCs (28 HBV‐related HCCs and 33 HCV‐related HCCs) and 59 matched noncancerous livers, and five normal livers. Among analyzed genes, preferential methylation in HBV‐related HCC was validated in one gene only. However, 15 genes were found to be methylated preferentially in HCV‐related HCC, which was independent from age. Hierarchical clustering of HCC using these genes stratified HCV‐related HCC as a cluster of frequently methylated samples. The 15 genes included genes inhibitory to cancer‐related signaling such as RAS/RAF/ERK and Wnt/β‐catenin pathways. Methylation of dual specificity phosphatase 4 (DUSP4), cytochrome P450, family 24, subfamily A, polypeptide 1 (CYP24A1), and natriuretic peptide receptor A (NPR1) significantly correlated with recurrence‐free survival. It was indicated that genes methylated preferentially in HCV‐related HCC exist, and that DNA methylation might play an important role in HCV‐related HCC by silencing cancer‐related pathway inhibitors, and might perhaps be useful as a prognostic marker. (Cancer Sci 2010)