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Background and Aim: Chronic hepatitis C virus (HCV) infection, long‐term alcohol use, cigarette smoking, and obesity are the major risk factors for hepatocellular carcinoma (HCC) in the United States, but the disease risk varies substantially among individuals with these factors, suggesting host susceptibility to and gene–environment interactions in HCC. To address genetic susceptibility to HCC, we conducted a genome‐wide association study (GWAS). Methods: Two case‐control studies on HCC were conducted in the United States. DNA samples were genotyped using the Illumian microarray chip with over 710 000 single nucleotide polymorphisms (SNPs). We compared these SNPs between 705 HCC cases and 1455 population controls for their associations with HCC and verified our findings in additional studies. Results: In this GWAS, we found that two SNPs were associated with HCC at P < 5E‐8 and six SNPs at P < 5E‐6 after adjusting for age, sex, and the top three principal components (PCs). Five of the SNPs in chromosome 22q13.31, three in PNPLA3 (rs2281135, rs2896019, and rs4823173) and two in SAMM50 (rs3761472, rs3827385), were replicated in a small US case‐control study and a cohort study in Singapore. The associations remained significant after adjusting for body mass index and HCV infection. Meta‐analysis of multiple datasets indicated that these SNPs were significantly associated with HCC. Conclusions: SNPs in PNPLA3 and SAMM50 are known risk loci for nonalcoholic fatty liver disease (NAFLD) and are suspected to be associated with HCC. Our GWAS demonstrated the associations of these SNPs with HCC in a US population. Biological mechanisms underlying the relationship remain to be elucidated. [ABSTRACT FROM AUTHOR]

The cancer stem cell marker, Ep CAM, is an important indicator of Wnt/β-catenin signaling activation and a functional component of hepatocellular tumor-initiating cells. A high-throughput screening assay was developed to identify inhibitors of Ep CAM-dependent growth of hepatocellular carcinoma (HCC) cells. Ep CAM(+) and Ep CAM(−) HCC cell lines were assessed for differential sensitivity to a Wnt/β-catenin pathway inhibitor. Libraries comprising 22 668 pure compounds and 107 741 crude or partially purified natural product extracts were tested, and 12 pure compounds and 67 natural product extracts were identified for further study. Three active compounds and the positive control were further characterized in terms of effects on Ep CAM expression. Treatment of Ep CAM(+) Hep3B cells resulted in loss of Ep CAM expression as assessed by flow cytometry. This reduction was incomplete (most cells continued to express Ep CAM), but resulted in generation of cell populations expressing lower levels of Ep CAM. Sublethal concentrations (~ IC50) reduced median Ep CAM expression to 28% of control after 1 day and 19% of control after 2 days. Reduction in Ep CAM expression preceded growth inhibition suggesting that a threshold of Ep CAM expression may be required for growth of Ep CAM-dependent cells. The identification of compounds with a variety of possible molecular targets suggests a likelihood of multiple mechanisms for modulation of Ep CAM-dependent cell growth. [ABSTRACT FROM AUTHOR]

Background A 17-cytokine gene expression signature in noncancerous hepatic tissue from patients with metastatic hepatocellular carcinoma (HCC) was recently found to predict HCC metastasis and recurrence. We examined whether the cytokine gene expression profile of noncancerous lung tissue could predict the meta- static capability of adjacent lung adenocarcinoma. Methods We analyzed a 15-cytokine gene expression profile in noncancerous lung tissue and corresponding lung tumor tissue from 80 US lung adenocarcinoma patients using real-time quantitative reverse transcription- polymerase chain reaction. We then used unsupervised hierarchical clustering and Prediction Analysis of Microarray classification to test the prognostic ability of the 15-cytokine gene profile in the US patients and in an independent validation set comprising 50 Japanese patients with stage I disease. Survival was analyzed by the Kaplan-Meier method using the log-rank test, and univariate and multivariable Cox proportional hazards modeling were used to analyze the association of clinical variables with patient survival. All statistical tests were two-sided. Results A 15-cytokine gene signature in noncancerous lung tissue primarily reflected the lymph node status of 80 lung adenocarcinoma patients, whereas the gene signature of the corresponding lung tumor tissue was associated with prognosis independent of lymph node status. Cytokine Lung Adenocarcinoma Survival Signature of ii genes (CLASS-11), a refined 11-gene signature, accurately classified patients, including those with stage I disease, according to risk of death from adenocarcinoma. CLASS-11 prognostic classification was statistically significantly associated with survival and was an independent prognostic factor for stage I patients (hazard ratio for death in the high-risk CLASS-11 group compared with the low-risk CLASS-11 reference group = 7.46, 95% confidence interval = 2.14 to 26.05; P= .002). CLASS-11 also classified patients in the validation set according to risk of recurrence. Conclusion CLASS-11, which consists of genes for pro- and anti-inflammatory cytokines, identifies stage I lung adenocarcinoma patients who have a poor prognosis. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is a fatal disease occurring worldwide and developing mainly in chronic liver diseased patients. Despite routine screening of individuals at high risk, most of the patients are diagnosed at late stages of HCC. In addition, the recurrence rate after surgical resection of small tumors is high. Molecular profiling, including expression analysis, comparative genomics and proteomics, provides powerful tools to gain insight into the molecular mechanisms underlying carcinogenesis. Advances in bioinformatics have also allowed for the evaluation of large data sets. Therefore, molecular profiling of HCC using a Biological Expression Network Discovery (BLEND) strategy that integrates global molecular profiling data, including mRNA, miRNA, DNA methylation and DNA copy numbers from both the tumor and the surrounding microenvironment, along with mechanistic studies, may improve the diagnosis, treatment and prognosis of HCC patients. Such an approach will provide mechanistic insight into the pathogenesis of HCC, potentially leading to personalized medicine and the identification of new therapeutic targets. [ABSTRACT FROM AUTHOR]

Summary: Hepatocellular carcinoma (HCC) is an aggressive malignancy mainly due to metastases or postsurgical recurrence. We postulate that metastases are influenced by the liver microenvironment. Here, we show that a unique inflammation/immune response-related signature is associated with noncancerous hepatic tissues from metastatic HCC patients. This signature is principally different from that of the tumor. A global Th1/Th2-like cytokine shift in the venous metastasis-associated liver microenvironment coincides with elevated expression of macrophage colony-stimulating factor (CSF1). Moreover, a refined 17 gene signature was validated as a superior predictor of HCC venous metastases in an independent cohort, when compared to other clinical prognostic parameters. We suggest that a predominant humoral cytokine profile occurs in the metastatic liver milieu and that a shift toward anti-inflammatory/immune-suppressive responses may promote HCC metastases. [Copyright &y& Elsevier]

The current metastasis paradigm suggests that the primary tumor starts off benign but over time slowly acquires changes that provide a few rare cells within the tumor the ability to metastasize. However, this concept has been challenged by several recent studies using the microarray-based approach. We have recently found that the molecular signature of primary hepatocellular carcinoma (HCC) is very similar to that of their corresponding metastases, while it differs significantly in primary HCCs with or without metastasis. Similar findings are also evident in primary cancers of the lung, breast, and prostate. Such a signature can be used to predict the prognosis of HCC patients. Moreover, there are significant differences in the gene expression profiles of liver parenchyma among HCC patients with or without intrahepatic metastases. These findings imply that many of the metastasis-promoting genes are embedded in the primary tumors and that the ability to metastasize may be an inherent quality of the tumor from the beginning. In addition, the condition of liver parenchyma may dictate the intrahepatic metastasis potential, which is consistent with the hypothesis that the degree of viral-hepatitis-mediated liver damage or possibly the genetic makeup of individuals may play an important role in metastasis. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Chronic inflammation and chromosome aneuploidy are major traits of primary liver cancer (PLC), which represent the second most common cause of cancer-related death worldwide. Increased cancer fitness and aggressiveness of PLC may be achieved by enhancing tumoral genomic complexity that alters tumor biology. Here, we developed a scoring method, namely functional genomic complexity (FGC), to determine the degree of molecular heterogeneity among 580 liver tumors with diverse ethnicities and etiologies by assessing integrated genomic and transcriptomic data. We found that tumors with higher FGC scores are associated with chromosome instability and TP53 mutations, and a worse prognosis, while tumors with lower FGC scores have elevated infiltrating lymphocytes and a better prognosis. These results indicate that FGC scores may serve as a surrogate to define genomic heterogeneity of PLC linked to chromosomal instability and evasion of immune surveillance. Our findings demonstrate an ability to define genomic heterogeneity and corresponding tumor biology of liver cancer based only on bulk genomic and transcriptomic data. Our data also provide a rationale for applying this approach to survey liver tumor immunity and to stratify patients for immune-based therapy. [ABSTRACT FROM AUTHOR]

Summary Intrahepatic cholangiocarcinoma (ICC) and hepatocellular carcinoma (HCC) are clinically disparate primary liver cancers with etiological and biological heterogeneity. We identified common molecular subtypes linked to similar prognosis among 199 Thai ICC and HCC patients through systems integration of genomics, transcriptomics, and metabolomics. While ICC and HCC share recurrently mutated genes, including TP53 , ARID1A , and ARID2 , mitotic checkpoint anomalies distinguish the C1 subtype with key drivers PLK1 and ECT2 , whereas the C2 subtype is linked to obesity, T cell infiltration, and bile acid metabolism. These molecular subtypes are found in 582 Asian, but less so in 265 Caucasian patients. Thus, Asian ICC and HCC, while clinically treated as separate entities, share common molecular subtypes with similar actionable drivers to improve precision therapy. [ABSTRACT FROM AUTHOR]

Background & Aims: We combined gene expression and metabolic profiling analyses to identify factors associated with outcomes of patients with hepatocellular carcinoma (HCC). Methods: We compared metabolic and gene expression patterns between paired tumor and nontumor tissues from 30 patients with HCC, and validated the results using samples from 356 patients with HCC. A total of 469 metabolites were measured using liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry. Metabolic and genomic data were integrated, and Kaplan–Meier and Cox proportional hazards analyses were used to associate specific patterns with patient outcomes. Associated factors were evaluated for their effects on cancer cells in vitro and tumor formation in nude mice. Results: We identified 28 metabolites and 169 genes associated with aggressive HCC. Lipid metabolites of stearoyl-CoA-desaturase (SCD) activity were associated with aberrant palmitate signaling in aggressive HCC samples. Expression of gene products associated with these metabolites, including SCD, were associated independently with survival times and tumor recurrence in the test and validation sets. Combined expression of SCD and α-fetoprotein were associated with outcomes of patients with early-stage HCC. Levels of monounsaturated palmitic acid, the product of SCD activity, were increased in aggressive HCCs; monounsaturated palmitic acid increased migration and invasion of cultured HCC cells and colony formation by HCC cells. HCC cells that expressed small interfering RNA against SCD had decreased cell migration and colony formation in culture and reduced tumorigenicity in mice. Conclusions: By using a combination of gene expression and metabolic profile analysis, we identified a lipogenic network that involves SCD and palmitate signaling and was associated with HCC progression and patient outcomes. The microarray platform and data have been submitted to the Gene Expression Omnibus public database at NCBI following MIAME guidelines. Accession numbers: GPL4700 (platform), and GSE6857 (samples). [Copyright &y& Elsevier]

Background & Aims: Hepatocellular carcinoma (HCC) is an aggressive malignancy; its mechanisms of development and progression are poorly understood. We used an integrative approach to identify HCC driver genes, defined as genes whose copy numbers associate with gene expression and cancer progression. Methods: We combined data from high-resolution, array-based comparative genomic hybridization and transcriptome analysis of HCC samples from 76 patients with hepatitis B virus infection with data on patient survival times. Candidate genes were functionally validated using in vitro and in vivo models. Results: Unsupervised analyses of array comparative genomic hybridization data associated loss of chromosome 8p with poor outcome (reduced survival time); somatic copy number alterations correlated with expression of 27.3% of genes analyzed. We associated expression levels of 10 of these genes with patient survival times in 2 independent cohorts (comprising 319 cases of HCC with mixed etiology) and 3 breast cancer cohorts (637 cases). Among the 10-gene signature, a cluster of 6 genes on 8p, (DLC1, CCDC25, ELP3, PROSC, SH2D4A, and SORBS3) were deleted in HCCs from patients with poor outcomes. In vitro and in vivo analyses indicated that the products of PROSC, SH2D4A, and SORBS3 have tumor-suppressive activities, along with the known tumor suppressor gene DLC1. Conclusions: We used an unbiased approach to identify 10 genes associated with HCC progression. These might be used in assisting diagnosis and to stage tumors based on gene expression patterns. [Copyright &y& Elsevier]

Background & Aims: Mutations in TP53, a tumor suppressor gene, are associated with prognosis of many cancers. However, the prognostic values of TP53 mutation sites are not known for patients with hepatocellular carcinoma (HCC) because of heterogeneity in their geographic and etiologic backgrounds. Methods: TP53 mutations were investigated in a total of 409 HCC patients, including Chinese (n = 336) and white (n = 73) patients, using the direct sequencing method. Results: A total of 125 TP53 mutations were found in Chinese patients with HCC (37.2%). HCC patients with TP53 mutations had a shorter overall survival time compared with patients with wild-type TP53 (hazard ratio [HR], 1.86; 95% confidence interval [CI]: 1.37–2.52; P < .001). The hot spot mutations R249S and V157F were significantly associated with worse prognosis in univariate (HR, 2.11; 95% CI: 1.51–2.94; P < .001) and multivariate analyses (HR, 1.79; 95% CI: 1.29–2.51; P < .001). Gene expression analysis revealed the existence of stem cell-like traits in tumors with TP53 mutations. These findings were validated in breast and lung tumor samples with TP53 mutations. Conclusions: TP53 mutations, particularly the hot spot mutations R249S and V157F, are associated with poor prognosis for patients with HCC. The acquisition of stem cell-like gene expression traits might contribute to the aggressive behavior of tumors with TP53 mutation. [ABSTRACT FROM AUTHOR]

Background & Aims: Hepatocellular carcinoma (HCC) is an aggressive cancer with a poor prognosis mainly due to metastasis. MicroRNAs are endogenous small noncoding RNAs that regulate cellular gene expression and are functionally linked to tumourigenesis. Using microarray analysis, we recently identified 20 miRNAs associated with HCC metastasis. Here, we carried out further analyses on one of these microRNAs, let-7g, to determine whether it is functionally linked to HCC metastasis. Methods: Quantitative real-time polymerase chain reaction was used to determine the level of mature let-7g transcript in HCC clinical specimens and its correlation with patient survival. Ectopic expression of let-7g was carried out in HCC cell lines to assess its influence on cell growth, migration, and invasion. Results: We confirmed that the level of let-7g was significantly lower in metastatic HCCs compared to metastasis-free HCCs. Moreover, low let-7g expression in a tumour was predictive of poor survival in HCC patients. Functional studies indicated that ectopic expression of let-7g significantly inhibits HCC cell migration and cell growth. In-silico analysis revealed members of soluble collagens as potential targets of let-7g. Consistently, the levels of type I collagen α2 (COL1A2) and let-7g were inversely correlated in HCC clinical specimens. COL1A2 was experimentally validated as a direct target of let-7g. Moreover, addition of COL1A2 counteracted the inhibitory effect of let-7g on cell migration. Conclusions: These results suggest that let-7g may suppress HCC metastasis partially through targeting COL1A2. [Copyright &y& Elsevier]

Background: Hepatocellular carcinoma is a common and aggressive cancer that occurs mainly in men. We examined microRNA expression patterns, survival, and response to interferon alfa in both men and women with the disease. Methods: We analyzed three independent cohorts that included a total of 455 patients with hepatocellular carcinoma who had undergone radical tumor resection between 1999 and 2003. MicroRNA-expression profiling was performed in a cohort of 241 patients with hepatocellular carcinoma to identify tumor-related microRNAs and determine their association with survival in men and women. In addition, to validate our findings, we used quantitative reverse-transcriptase–polymerase-chain-reaction assays to measure microRNAs and assess their association with survival and response to therapy with interferon alfa in 214 patients from two independent, prospective, randomized, controlled trials of adjuvant interferon therapy. Results: In patients with hepatocellular carcinoma, the expression of miR-26a and miR-26b in nontumor liver tissue was higher in women than in men. Tumors had reduced levels of miR-26 expression, as compared with paired noncancerous tissues, which indicated that the level of miR-26 expression was also associated with hepatocellular carcinoma. Moreover, tumors with reduced miR-26 expression had a distinct transcriptomic pattern, and analyses of gene networks revealed that activation of signaling pathways between nuclear factor κB and interleukin-6 might play a role in tumor development. Patients whose tumors had low miR-26 expression had shorter overall survival but a better response to interferon therapy than did patients whose tumors had high expression of the microRNA. Conclusions: The expression patterns of microRNAs in liver tissue differ between men and women with hepatocellular carcinoma. The miR-26 expression status of such patients is associated with survival and response to adjuvant therapy with interferon alfa. N Engl J Med 2009;361:1437-47. [ABSTRACT FROM AUTHOR]