MT1M and MT1G promoter methylation as biomarkers for hepatocellular carcinoma

AIM:To investigate the potential of promoter methylation of two tumor suppressor genes(TSGs)as biomarkers for hepatocellular carcinoma(HCC).METHODS:A total of 189 subjects were included in this retrospective cohort,which contained 121 HCC patients without any history of curative treatment,37 patients with chronic hepatitis B(CHB),and 31 normal controls(NCs).DNA samples were extracted from 400μL of serum of each subject and then modified using bisulfite treatment.Methylation of the promoters of the TSGs(metallothionein1M,MT1M;and metallothionein 1G,MT1G)was determined using methylation-specific polymerase chain reaction.The diagnostic value of combined MT1M and MT1G promoter methylation was evaluated using the area under the receiver operating characteristic curves.RESULTS:Our results indicated that the methylation status of serum MT1M(48.8%,59/121)and MT1G(70.2%,85/121)promoters in the HCC group was significantly higher than that in the CHB group(MT1M 5.4%,2/37,P<0.001;MT1G 16.2%,6/37,P<0.001)and NC group(MT1M 6.5%,2/31,P<0.001;MT1G 12.9%,4/27,P<0.001).Aberrant serum MT1M promoter methylation gave higher specificity to discriminate HCC from CHB(94.6%)and NCs(93.5%),whereas combined methylation of serum MT1M and MT1G promoters showed higher diagnostic sensitivity(90.9%),suggesting that they are potential markers for noninvasive detection of HCC.Furthermore,MT1M promoter methylation was positively correlated with tumor size(rs=0.321,P<0.001),and HCC patients with both MT1M and MT1G promoter methylation tended to show a higher incidence of vascular invasion or metastasis(P=0.018).CONCLUSION:MT1M and MT1G promoter methylation may be used as serum biomarkers for noninvasive detection of HCC.     

Oncogenic Wnt3a expression as an estimable prognostic marker for hepatocellular carcinoma

AIM: To investigate member 3a of Wingless-type MMTV integration site family(Wnt3a) expression in cancerous and surrounding tissues and the relationship between clinicopathologic features of hepatocellular carcinoma(HCC) and Wnt3 a expression.METHODS: Wnt3 a expression and cellular distribution and clinicopathologic characteristics in cancerous tissue and matched surrounding tissues were analyzed in 80 HCC patients from January 2006 to August 2008 by tissue microarrays and immunohistochemistry. The overall and disease-free survival rates were estimated using the Kaplan-Meier method and compared with the log-rank test. The prognostic analysis was carried out with univariate and multivariate Cox regressions models.RESULTS : The incidence of oncogenic Wnt3a expression in the cancerous group was up to 96.25%(77 of 80), which was significantly higher(χ2 = 48.818, P 0.001) than that in the surrounding group(46.25%, 37 of 80). Brown Wnt3 a staining gradually increased with clinical staging that showed very strong staining in advanced HCC. The clinicopathologic features of high Wnt3 a expression in HCC were related to poorlydifferentiated grade(χ2 = 20.211, P 0.001), liver cirrhosis(χ2 = 8.467, P 0.004), hepatitis B virus(HBV) infection(χ2 = 12.957, P 0.001), higher tumor-nodemetastasis stage(χ2 = 22.960, P 0.001), and 5-year survival rate(χ2 = 15.469, P 0.001).CONCLUSION: Oncogenic Wnt3 a expression associated with HBV infection and cirrhotic liver might be an independent prognostic factor for HCC.     

Aristaless-like homeobox-4 gene methylation is a potential marker for colorectal adenocarcinomas

BACKGROUND & AIMS: The identification of novel genetic and epigenetic markers indicative of changes in the pathogenesis of colon cancer, along with easier-to-use, more sensitive assay methods, may improve the detection, treatment, and overall prognosis of this malignancy. METHODS: Using methylation-specific arbitrarily primed polymerase chain reaction, a fragment of the Aristaless-like homeobox-4 (ALX4) gene that was highly methylated in colon adenomas and cancer was identified. Methylation of ALX4 was analyzed in colorectal adenomas and cancers, in the liver metastases of patients with colorectal cancer, and in 61 other neoplasias, including gastric, esophageal, and hepatocellular cancer and cholangiocarcinoma. ALX4 methylation was also analyzed in the serum of 30 patients with colon cancer. RESULTS: ALX4 gene methylation was confirmed in colon adenomas (11/13) and more frequently present in primary colorectal cancers (30/47) compared with the normal colon mucosa (0/21) (P < .0001). In addition, ALX4 methylation was frequently observed in adenocarcinomas of the esophagus (12/14), stomach (11/15), and bile ducts (4/5) compared with all other cancers (P < .001). ALX4 gene methylation was also more frequently found in sera of patients with colon cancer compared with noncancer controls (P < .0001). Using a cutoff of 41.4 pg/mL, sensitivity and specificity were 83.3% and 70%, respectively. CONCLUSIONS: Apart from colon adenomas and primary and metastatic colorectal cancers, ALX4 is frequently methylated in adenocarcinomas of the gastrointestinal tract. ALX4 gene methylation in sera of patients with cancer may thus serve as a methylation-specific test for colon and other gastrointestinal cancers.     

Alpha-fetoprotein expression is a potential prognostic marker in hepatocellular carcinoma

AIM: To characterize the alpha-fetoprotein (AFP) positive and negative hepatocellular carcinoma (HCC) samples. METHODS: Thirty-seven paraffin-embedded human HCC samples were analyzed by immunohistochemistry for the following antigens: AFP, β-catenin, p53, CD44, MSH-2, MLH-1, and HNF-4. The tumors were divided into two groups based on the AFP expression. The immunophenotypic data and important clinical parameters were studied between the two groups. RESULTS: Twenty-one of the thirty-seven examined HCCs were AFP positive. Seven with nuclear p53 staining were AFP positive, while seven tumors with nuclear β-catenin staining were AFP negative. CD44 staining and high histological tumor grade were more frequent among the AFP-positive HCCs. The other immunophenotypical and dinical parameters did not show statistically significant difference in their distribution between the AFP positive and negative samples. CONCLUSION: AFP expression in HCC correlates with unfavorable prognostic factors, while nuclear β-catenin positivity is more common among the AFP-negative liver tumors. This observation supports the microarray data on in vivo human tumors.     

Methylation of Dickkopf-3 as a prognostic factor in cirrhosis-related hepatocellular carcinoma

AIM:To investigate the prevalence and time of Dickkopf(DKK) family methylation and its clinical significance in hepatocarcinogenesis. METHODS:Methylation of DKK family genes was quantitatively analyzed in 115 liver tissue samples,including 50 pairs of primary hepatocellular carcinoma(HCC) and matched noncancerous cirrhotic tissue samples,as well as 15 liver cirrhosis biopsy samples. RESULTS:The methylation level of DKK3 was significantly higher in HCC tissue samples than in matched noncancerous cirrhotic ti...     

反向聚合酶链反应检测肝细胞癌中乙型肝炎病毒DNA整合

目的 应用反向聚合酶链反应(inverse polymerase chain reaction,IPCR)法检测肝细胞癌(HCC)中乙型肝炎病毒(HBV)DNA整合。方法 提取手术切除石蜡包埋入肝癌组织中DNA,根据IPCR原理,选用HBV DRl区无酶切位点的限制性内切酶酶切,通过连接反应形成环化DNA分子,以此作为模板进行PCR扩增得到已知序列的旁侧序列。琼脂糖凝胶电泳观察PCR扩增产物片段。结果 19例HCC标本中有14例检出整合型HBV DNA,2例同时存在游离型HBV DNA。结论 IPCR可以准确测定肝细胞中HBVDNA的整合。该方法为研究HBV DNA在肝细胞中的整合机制提供一简单、快速、经济途径。     

Methylation status of suppressor of cytokine signaling-1 gene in hepatocellular carcinoma

Background Silencing of the suppressor of cytokine signaling (SOCS-1) by aberrant methylation at the CpG island in the coding region gene has been reported in hepatocellular carcinoma (HCC). However, principally, it is methylation in the 5-noncoding region but not that in the coding region which determines the regulation of gene expression.Methods Methylation-specific PCR was performed for the analysis of methylation status both in the 5-noncoding region and the CpG island of SOCS-1 from 22 HCC tissue samples with adjacent non-HCC tissue samples and from two cell lines.Results Using primers in the CpG island, 9 of 22 HCC samples exhibited aberrant methylation of SOCS-1, while only 1 of 22 adjacent non-HCC samples did so. The unmethylation pattern was detected in 1 of 22 HCC and in 5 of 22 non-HCC samples. Thus, aberrant methylation of SOCS-1 was significantly associated with HCC (P = 0.0076 by Fishers exact test). Using primers in the 5-noncoding region, aberrant methylation was observed in 12 of 22 HCC and in 2 non-HCC samples. The unmethylated pattern was observed in 5 of 22 HCC and in 10 of 22 non-HCC samples (P = 0.0042). There was no significant correlation between the methylation status of SOCS-1 and clinicopathological findings, such as the presence or absence of cirrhosis or the histological grade of HCC.Conclusions Aberrant methylation of the SOCS-1 had a significant correlation with HCC. The rate of aberrant methylation was similar in the 5-noncoding region and in the CpG island. Aberrant methylation of SOCS-1 may be associated with hepatocarcinogenesis, although further studies are necessary.     

Glypican-3 is a prognostic factor and an immunotherapeutic target in hepatocellular carcinoma

Glypican-3 (GPC3) is a cell surface oncofetal proteoglycan that is anchored by glycosylphosphatidylinositol. Whereas GPC3 is abundant in fetal liver, its expression is hardly detectable in adult liver. Importantly, GPC3 is overexpressed in hepatocellular carcinoma (HCC), and several immunohistochemical studies reported that overexpression predicts a poorer prognosis for HCC patients. Therefore, GPC3 would serve as a useful molecular marker for HCC diagnosis and also as a target for therapeutic intervention in HCC. Indeed, some immunotherapy protocols targeting GPC3 are under investigations; those include humanized anti-GPC3 cytotoxic antibody, peptide vaccine and immunotoxin therapies. When considering the clinical requirements for GPC3-targeting therapy, companion diagnostics to select the appropriate HCC patients are critical, and both immunohistochemical analysis of tissue sections and measurement of serum GPC3 level have been suggested for this purpose. This review summarizes current knowledge regarding the clinical implication of GPC3 detection and targeting in the management of patients with HCC.     

A polymorphism within ErbB4 is associated with risk for hepatocellular carcinoma in Chinese population

AIM: To investigate the association between hepatocellular carcinoma (HCC) susceptibility and a 12-bp insertion/deletion polymorphism (rs6147150) in the 3’UTR of ErbB4.METHODS: Using a case-control design, the rs6147150 genotypes in 270 patients with HCC and 270 healthy controls were determined by direct polymerase chain reaction and polyacrylamide gel electrophoresis. Logistic regression was used to analyze the association between the polymorphism and cancer risk.RESULTS: Computational modeling suggested that rs6147150 was located in the seed region of hsa-let-7c, a potential target sequence in ErbB4 3’UTR. Logistic regression analysis showed that, compared with individuals homozygous for wild-type, heterozygotes [adjusted odds ratio (OR) = 1.48, 95% confidence interval (CI) = 1.03-2.17, P = 0.034] and individuals homozygous for 12-bp del/del (OR = 2.50, 95% CI = 1.37-4.56, P = 0.001) were at significantly higher risk of HCC. Carriers of the “del” allele of rs6147150 had a 1.59-fold increased risk for HCC (95% CI = 1.22-2.07, P = 0.003).CONCLUSION: rs6147150 may be associated with HCC risk, in part through let-7c-mediated regulation, and may be involved in the pathogenesis of HCC in Chinese populations.     

Allelic loss on chromosome 5q34 is associated with poor prognosis in hepatocellular carcinoma

PURPOSE: To identify and characterize novel genetic alterations in hepatocellular carcinoma (HCC). METHODS: DNA was extracted from 29 HCC and corresponding normal tissues and amplified with 59 different 10-base arbitrary primers. A 550 bp DNA fragment amplified using primer Q-9 and which was present in 19 of 29 cases (66%) was cloned, sequenced, and compared with known nucleotide sequences deposited in Genome database, and quantified by real-time PCR. RESULTS: DNA alterations were found on chromosomes 5q34, 6p25.2 and 8q12.1 in 11 of 29 cases (38%), 7 of 29 cases (24%), and 12 of 29 cases (41%), respectively. Multivariate analysis showed that the allelic loss on chromosome 5q34 was an independent prognostic factor for poor survival of HCC patients, with the median survival time of 19 weeks for allelic loss versus 109 weeks for no allelic loss (P = 0.001). CONCLUSIONS: This study indicates that allelic loss on chromosome 5q34 may be involved in the development of HCC and could be used as a prognostic indicator in HCC patients.     

肝细胞肝癌谷胱甘肽硫转移酶P1甲基化研究

目的本研究探讨谷胱甘肽硫转移酶（GST）PI启动子CpG岛甲基化与肝细胞肝癌（HCC）的关系。方法用甲基化特异性PCR技术检测53例HCC肿瘤组织及其癌旁非肿瘤组织中，GSTP1基因启动子CpG岛甲基化状况。结果GSTP1基因在HCC肿瘤组织中的甲基化率显著高于癌旁非肿瘤组织（X^2=19．08，P〈0．001），在Ⅲ-Ⅳ期肿瘤中的甲基化率显著高于Ⅰ-Ⅱ期肿瘤（X^2=4．84，P=0．028），在不同大小的肿瘤之间、单个结节与多个结节的肿瘤之间以及包膜完整的肿瘤与包膜不完整的肿瘤之间甲基化率的差异均无显著性。结论GSTP1启动子CDG岛甲基化可能参与肝细胞的痛性转变。     

MRGBP is a potential novel prognostic biomarker and is correlated with immune infiltrates in hepatocellular carcinoma

This study investigated the expression change, prognostic values, and potential regulatory mechanisms of mortality factor on chromosome 4 (MORF4)-related gene-binding protein (MRGBP) in hepatocellular carcinoma (HCC).MRGBP expression and clinical data from The Cancer Genome Atlas were used to evaluate the associations between MRGBP expression and clinicopathological characteristics. Kaplan–Meier and Cox regression analyses were performed to assess the factors contributing to prognosis. Gene set enrichment analysis (GSEA) was used to identify pathways associated with MRGBP expression. Single-sample gene set enrichment analysis (ssGSEA) was used to comprehensively analyze the relative immune infiltration levels.High MRGBP expression was significantly associated with a higher T stage, pathologic stage, histologic grade, vascular invasion, tumor protein p53 status, and worse overall survival. MRGBP exhibited high diagnostic accuracy with an area under the receiver operating characteristic curve value of 0.980. GSEA revealed the enrichment of pathways related to tumorigenesis in the MRGBP high-expression phenotype, such as cell cycle and DNA replication pathways. ssGSEA revealed that MRGBP expression was significantly correlated with 15 types of immune cell infiltration levels. The Wilcoxon rank sum test revealed significantly high T helper (Th), T follicular helper, CD56 bright natural killer, and Th2 cell enrichment scores in the high MRGBP expression group and significantly low neutrophil, Th17, dendritic cell (DC), gamma delta T, cytotoxic cell, regulatory T cell, plasmacytoid DC, and immature DC enrichment scores.MRGBP may be a novel prognostic biomarker and a therapeutic target correlated with immune infiltrates in HCC.     

Diagnostic value of glypican-3 in serum and liver for primary hepatocellular carcinoma

AIM:To evaluate the diagnostic value of glypican-3(GPC3) in serum and liver for primary hepatocellular carcinoma(HCC).METHODS:Serum levels of GPC3 and α-fetoprotein(AFP) were measured in 75 patients with primary HCC and 32 patients with liver cirrhosis.Expression of GPC3 and AFP in 58 HCC and 12 cirrhotic specimens was detected with immunohistochemical staining.RESULTS:When the cut-off value of serum GPC3 was set at 300 ng/L,its sensitivity and specificity for HCC were 47.0% and 93.5%,respectively.Among the...     

POLD1基因在原发性肝癌中的表达及其意义

目的:探讨DNA聚合酶δ催化亚基基因(DNA polymerase delta catalytic subunit gene 1,POLD1)及其编码蛋白p125在原发性肝癌中的表达及意义.方法:选用原发性肝癌患者手术切除标本20例,分别采用逆转录聚合酶链反应(RT-PCR)法和Western blot法检测POLD1...