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The patients with lymph node metastasis from hepatocellular carcinoma may survive only a few months. These patients are not suitable for transcatheter arterial chemoembolization, percutaneous ethanol injection and surgical resection. External beam radiation therapy (EBRT) could provide a useful treatment, but only 4 hepatocellular carcinoma cases with abdominal lymph nodes involvement were treated with EBRT in the two reports over the last 10 years. In this paper, we report 29 hepatocellular carcinoma patients with abdominal lymph node metastasis who received EBRT over a period of 5 years.Of 29 patients, 20 were pathologically confirmed to have hepatocellular carcinoma and 9 patients were clinically diagnosed to have hepatocellular carcinoma. All of the patients had proven abdominal lymph node metastasis by enhanced abdominal computer tomography scan due to clinical follow-up or abdominal symptoms. EBRT was not the initial treatment in all. The patients received locoregional lymph nodes irradiation. The tumor dose ranged from 30 to 60 Grays (Gy) in daily 2.0-Gy fractions (Fx), 5 times a week.It is of interest to note that an objective regression (complete response and partial response) rate was 100%. The clinical symptoms were relieved. The median survival time was 8 (4-28) months. The overall 1-year and 2-year survival rates were 43.5% and 10.5%, respectively. The median survival time consistently decreased as the lymph node involvement increase follows the natural flow of lymph.Lymph node metastasis from hepatocellular carcinoma is sensitive to EBRT, although EBRT is palliative in intent, it is useful in the treatment with 50 Gy/25 Fx for those patients.

Recurrence and metastasis in hepatocellular carcinoma remains a major challenge to further improve survival. High frequency of loss of heterozygosity at D14S62 and D14S51 in tumor tissue has been shown to be closely related to metastasis and recurrence in breast cancer. But, loss of heterozygosity on 14q in plasma and tumor tissue DNA of hepatocellular carcinoma patients has not been investigated. To establish a way to predict metastasis and recurrence after curative hepatic resection, we analyzed loss of heterozygosity on 14 q in plasma and tumor tissue DNA of hepatocellular carcinoma patients with curative resection.We used a simple, rapid and non-radioactive method to analyze loss of heterozygosity at D14S62 and D14S51 in paired plasma, lymphocyte and tumor tissue DNA of 85 hepatocellular carcinoma patients with curative resection.From 79 cases informative for D14S62 and 78 cases informative for D14S51 of 85 hepatocellular carcinoma tissue DNA, loss of heterozygosity at D14S62 and D14S51 was present in 45 (57.0%) and 41 (52.6%) cases respectively. And in 96.0% of the tissues which showed loss of heterozygosity we were able to detect loss of heterozygosity in their matched plasma. In matched 85 cases of hepatocellular carcinoma plasma DNA, we detected loss of heterozygosity at D14S62 in 55.7% and at D14S51 in 50.0% of the respective informative DNA samples. The loss of heterozygosity patterns of plasma DNA were almost identical to their corresponding tumor tissues. A comparison of these genetic changes with clinicopathological data of these checked hepatocellular carcinoma patients showed that loss of heterozygosity at D14S62 and D14S51 was adversely correlated significantly with the presence of tumor size, with 35.4% at both the D14S62 and D14S51 locus in the HTMR (high-tendency to metastasis and recurrence) group compared with 72.9% and 59.4% in the LTMR (low-tendency to metastasis and recurrence) group at D14S62 and D14S51, respectively (P = 0.001 and P = 0.027, respectively).Our results suggest that loss of heterozygosity at D14S62 and D14S51 plays an important role in the metastasis and recurrence of hepatocellular carcinoma patients following curative resection. Loss of heterozygosity at D14S62 and D14S51 in the plasma DNA of hepatocellular carcinoma patients detected by a simple and non-radioactive method has great potentials to be clinically used to predicate metastasis and recurrence after curative hepatic resection.

The authors' previous study suggested there could be metastasis suppressor gene for liver cancer on human chromosome 8p. TEY1 was a newly discovered metastasis suppressor gene for prostate cancer located on human chromosome 8p. In order to explore whether TEY1 gene could be a metastasis suppressor gene for liver cancer, the expression of TEY1 was quantitated in liver cancer cell strains with different metastatic potential.RNA was extracted from liver cancer cell strains, MHCC97-H, MHCC97-L, and HCCLM3, and normal liver cell line L02. The first strand cDNA was synthesized by reverse transcription, which was then used as the template to perform fluorescent quantitative polymerase chain reaction (FQ-PCR). The quantity of the expression of TEY1 gene was normalized by dividing with that of the housekeeping gene, GAPDH for each sample. One-way analysis of variance (ANOVA) was performed for the normalized values.The normalized initial cDNA concentrations of TEY1 in MHCC97-H, MHCC97-L, HCCLM3, and L02 were (3.57+/-2.62) x 10(-4), (5.02+/-1.95) x 10(-4), (2.82+/-0.78) x 10(-5), and (15.20+/-0.58) x 10(-4), respectively. The expression level of TEY1 in L02 was significantly different from those of liver cancer strains (P0.001). The expression level of TEY1 in HCCLM3 was also significantly different from those in MHCC97-H and MHCC97-L (P0.05). But there was no significant difference between MHCC97-H and MHCC97-L although the expression level of TEY1 gene in MHCC97-L was 1.4 times higher than that in MHCC97-H.The expression levels of TEY1 gene in liver cancer cells were negatively correlated with their different metastatic potentials; it could be conjectured that TEY1 may possibly be a metastasis suppressor gene for liver cancer.

The aim of this study was to examine the mechanism of interferon alpha (IFN-alpha) on inhibition of metastasis and recurrence of hepatocellular carcinoma (HCC). Nude mice bearing human HCC xenografts with high metastatic potential (LCI-D20) underwent curative resection of tumors on postimplant day 11. IFN-alpha was begun the next day at different dosages given subcutaneously for 35 consecutive days; normal saline solution was injected into the control mice. The mice were killed 48 hours after the final treatment, and the parameters were evaluated. The HCC intrahepatic recurrence rate, the size of the recurrent lesions, the rate of lung metastasis, the serum vascular endothelial growth factor level, and the microvessel density (immunohistochemistry) were as follows: 100%, 2136+/-794 mm(3)(mean+/-standard deviation), 100%, 265.7+/-154.7 pg/ml, and 144+/-37/HP, respectively, in the control mice, whereas these same values were 62.5%, 89+/-45 mm(3), 12.5%, 53.3+/-9.9 pg/ml, and 86+/-25/HP, respectively, in the IFN-alpha 1.5 x 10(7)U/kg treatment group (P

AIM: Our previous research on the surgical samples of primary liver cancer with CGH showed that the loss of human chromosome 8p had correlation with the metastatic phenotype of liver cancer. In order to seek the functional evidence that there could be a metastatsis suppressor gene (s) for liver cancer on human chromosome 8, we tried to transfer normal human chromosome 8 into rat liver cancer cell line C5F, which had high metastatic potential to lung. METHODS: Human chromosome 8 randomly marked with neo gene was introduced into C5F cell line by MMCT and positive microcell hybrids were screened by double selections of G418 and HAT. Single cell isolation cloning was applied to clone microcell hybrids. Finally, STS-PCR and WCP-FISH were used to confirm the introduction. RESULTS: Microcell hybrids resistant to HAT and G418 were obtained and 15 clones were obtained by single-cell isolation cloning. STS-PCR and WCP-FISH proved that human chromosome 8 had been successfully introduced into rat liver cancer cell line C5F. STS-PCR detected a random loss in the chromosome introduced and WCP-FISH found a consistent recombination of the introduced human chromosome with the rat chromosome. CONCLUSION: The successful introduction of human chromosome 8 into highly metastatic rat liver cancer cell line builds the basis for seeking functional evidence of a metastasis suppressor gene for liver cancer harboring on human chromosome 8 and its subsequent cloning.

Two hepatocellular carcinoma (HCC) cell clones named MHCC97-H and MHCC97-L with different metastatic potential have recently been established from the same parent cell line MHCC97 in our institute. The cytogenetic alterations of these two clones were investigated in this study to explore the possible clues to the mechanism involved in HCC metastasis.Their chromosomal aberrations were analyzed with comparative genomic hybridization (CGH), chromosome-specific painting, and two-color fluorescence in situ hybridization (FISH).The aberrations were found in a total of 17 chromosomes, and six kinds of the aberrations including gains of 1q, 7q, 8q, 20, and the losses of 8p23, 21q were found both in the two cell clones and their parent cell line MHCC97. Using modified CGH, with the DNA of MHCC97-L as control to test the MHCC97-H clone, the loss of 8p23 and the gain of 1q31-32, 8q21.3-22, 13q22, 17q22 were highlighted, and the most significant finding was on chromosome 8. Dual color FISH combining a pericentromeric probe and a BAC probe mapping at 8q23.1 was then performed to verify this result, and the signal ratios of the BAC to centromere were 1.43 in MHCC97-H and 1.45 in MHCC97-L, confirming the over-representations at 8q in both cells. Another interesting finding in the dual-color metaphase FISH was the intrachromosomal translocation of 8q to 8p (looked like an isochromosome 8) and non-reciprocal translocation of part of 8q to 4q, which was further clarified and proved by the FISH with whole chromosome 8 painting probe.The high copies amplification on 8q, the formation of isochromosome 8, non-reciprocal translocation of partial 8q to 4q, and loss of 8p occurred at the same time and are the characteristic chromosomal aberrations of the two cell clones. The chromosome 8p, especially 8p23, might harbor some novel genes related to the HCC metastasis.

The central role of sequential accumulation of genetic alterations during the development of cancer has been firmly established since the pioneering cytogenetic studies successfully defined recurrent chromosome changes in specific types of tumor. In the course of carcinogenesis, cells experience several genetic alterations that are associated with the transition from a preneoplastic lesion to an invasive tumor and finally to the metastatic state. Tumor progression is characterized by stepwise accumulation of genetic alterations. So does the dominant metastatic clone. Modern molecular genetic analyses have clarified that genomic changes accumulate during the development and progression of cancers. In comparison with the corresponding primary tumor, additional events of chromosomal aberrations (including gains or allelic losses) are frequently found in metastases, and the incidence of combined chromosomal alterations in the primary tumor, plus the occurrence of additional aberrations in the distant metastases, correlated significantly with decreased postmetastatic survival. The deletions at 3p, 4p, 6q, 8p, 10q, 11p, 11q, 12p, 13q, 16q, 17p, 18q, 21q, and 22q, as well as the over-representations at 1q, 8q, 9q, 14q and 15q, have been found to associate preferentially with the metastatic phenotype of human cancers. Among of them, the deletions on chromosomes 8p, 17p, 11p and 13p seem to be more significant, and more detail fine regions of them, including 8p11, 8p21-12, 8p22, 8p23, 17p13.3, 11p15.5, and 13q12-13 have been suggested harboring metastasis-suppressor genes. During the past decade, several human chromosomes have been functionally tested through the use of microcell-mediated chromosome transfer (MMCT), and metastasis-suppressor activities have been reported on chromosomes 1, 6, 7, 8, 10, 11, 12, 16, and 17. However, it is not actually known at what stage of the metastatic cascade these alterations have occurred. There is still controversial with the association between the chromosomal aberrations and the metastatic phenotype of cancer. As the progression of human genome project and the establishment of more and more new techniques, it is hopeful to make clear the genetic mechanisms involved in the tumor metastasis in a not very long future, and provide new clues to predicting and controlling the metastasis.

To understand the molecular mechanisms of metastasis in hepatocellular carcinoma (HCC), it is necessary to identify the accumulating genetic alterations during its progression as well as those responsible for the acquisition of metastatic potential in cancer cells. In our previous study, using comparative genomic hybridization (CGH), we found that loss on chromosome 8p is more frequent in metastatic lesions than in matched primary tumors of HCC. Thus, 8p deletion might contribute to HCC metastasis. To narrow the location of metastasis-related alteration regions, we analyzed 22 primary and matched metastatic lesions of HCC by genome-wide microsatellite analysis. Common regions with high levels of allelic imbalance (AI) were identified on 17p, 8p11-cen, 8p21-23, 4q32-qter, 4q13-23, 16q, and 1p33. Regions with increased AI in metastatic lesions were 8p23.3, 8p11.2, 17p11.2-13.3, 4q21-22, 4q32-qter, 8q24.1, 9p11, 9q31, 11q23.1, 13q14.1-31, 13q32-qter, 16p13.3, 16q13, 16q22, and 19p13.1, and these were considered to be related to the metastasis phenotype. Among them, loss on 8p was again proved to be related to progression and metastasis of HCC, and 8p23.3 and 8p11.2 were two likely regions harboring metastasis-related genes. It was also shown for the first time in HCC that AI of 19p13.1 might also be related to metastatic potential. These results provide some candidate regions for further study to identify putative genes suppressing metastasis of HCC.

It has previously been observed in animal studies that, at equivalent doses, radioimmunotherapy (RIT) is 2.5 times more effective than multiple fractions of external beam radiation therapy (EBRT) in inhibiting tumour growth. In this study, we compared the use of RIT and EBRT in patients with hepatocellular carcinoma (HCC), treated during the past 10 years. Of 67 patients without extrahepatic involvement, 32 were treated with hepatic artery ligation combined with RIT (the RIT group) while 35 were treated with a combination of hepatic arterial chemo-embolisation and EBRT (the EBRT group). The patients in the RIT group received (131)I-Hepama-1 monoclonal antibody, which was infused through the hepatic artery catheter. The patients in the EBRT group received transcatheter arterial chemo-embolisation and limited-field EBRT using a linear accelerator. Parameters observed include tumour response, alpha-fetoprotein (AFP) level in serum, human anti-murine antibody (HAMA) assay, T lymphocyte subsets, survival rates, routine parameters, sequential resection rates and histopathological status of the resection specimens. The sequential resection rates were 53% (17/32) and 23% (8/35), and tumour response rates were 72% (23/32) and 86% (30/35) in the RIT and EBRT groups, respectively. The main side-effects in the RIT group were mild allergic reactions. The most common toxicity in the EBRT group was an increase in liver enzymes. The liver tissue in the target volume was injured by EBRT. The injured liver tissue revealed a low-attenuation area adjacent to the hepatic tumour within the target volume on follow-up computed tomography studies after EBRT. On pathological evaluation, the low-attenuation area revealed hyperaemia, distended hepatic sinusoids packed with erythrocytes and hepatic cell loss. The sequential resection specimens from both the RIT and the EBRT group showed residual cancer tissue located at the edge of the mass. The residual cancer cells presented as giant cells under microscopy. T lymphocyte subsets observed prior to treatment did not significantly change after RIT, but were significantly disturbed by EBRT. HAMA formation was the major reason for discontinuing RIT, the incidence being as high as 34% (11/32). Intrahepatic and pulmonary metastases occurred more frequently in the EBRT group (63%) than in the RIT group (22%). The 1-, 2-, 3- and 4-year survival rates were 50%, 41%, 34% and 31% in the RIT group, and 77%, 39%, 11% and 7% in the EBRT group, respectively. It is interesting that the serum AFP level showed a transient increase, the mechanism and importance of which are not known, but are discussed. Both RIT and EBRT are useful treatment modalities for unresectable HCC, serving to prolong survival. However, RIT is much less toxic than EBRT, the side-effects of which include radiation injury to the liver and disturbance of T lymphocyte subsets.

Globally, hepatocellular carcinoma (HCC) accounts for 70-85% of primary liver cancers and ranks second in the leading cause of male cancer death. Serum alpha-fetoprotein (AFP), normally highly expressed in the liver only during fetal development, is reactivated in 60% of HCC tumors and associated with poor patient outcome. We hypothesize that AFP+ and AFP- tumors differ biologically. Using microarray-based global microRNA profiling in 223 HCC patients, we found that members of the miR-29 family were the most significantly (p Citation Format: Sonya T. Parpart, Stephanie Roessler, Fei Dong, Vinay Rao, Junfang Ji, Lun–Xiu Qin, Qing–Hai Ye, Zhao–You Tang, Hu–Liang Jia, Xin Wei Wang. Modulation of miR-29 expression by alpha-fetoprotein is linked to the hepatocellular carcinoma epigenome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5183. doi:10.1158/1538-7445.AM2014-5183

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).

Our previous studies demonstrated that osteopontin (OPN) plays a crucial role in hepatocellular carcinoma (HCC) metastasis. However, its function in epithelial-mesenchymal transition (EMT) remains obscure. In this study, we reported the critical role of OPN in EMT mediated HCC metastasis. Increased expression of OPN in human HCC predicts poor survival and disease recurrence after surgery. HCC tumors overexpressing OPN were featured by active EMT and motility/invasion of tumor cells both in vitro and in vivo, whereas silencing OPN expression attenuated EMT. OPN overexpression enhanced twist through activation of Akt, a critical EMT regulation of transcription factor. Suppression of the PI3K-AKT-Twist pathway interfered with OPN-mediated EMT and invasion. Consistently, we have observed a significant correlation between OPN expression and twist levels in a cohort of HCC patients, and found that combination of these two parameters is a more powerful predictor of poor prognosis. Conclusions: OPN plays a pivotal role in EMT mediated HCC metastasis and may serve as a novel prognostic biomarker and therapeutic target. Citation Format: Qiongzhu Dong, Xu-Chao Zhu, Chun Dai, Xiao-Fei Zhang, Hu-Liang Jia, Qing-Hai Ye, Lun-Xiu Qin. Osteopontin regulated epithelial-mesenchymal transition via PI3K/AKT signaling pathway in hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2695. doi:10.1158/1538-7445.AM2013-2695

To determine the state of CDKN2(p16/MTS1) gene in human hepatocellular carcinoma (HCC), the polymerase chain reaction (PCR), dot blot hybridization, PCR-single-strand conformation polymorphism (SSCP) and DNA sequencing techniques were applied to investigate the homozygous deletions and intragenic mutations of CDKN2 exon 2 in tumor and non-tumor liver tissues from 24 patients with HCCs. Alterations of CDKN2 exon 2 were found in 10 (2 homozygous deletions and 8 intragenic mutations) of the 24 tumor samples (41.7%), no alteration was found in any of the non-tumor liver tissues. All of the intragenic mutations were located at codon 81 of CDKN2 with GC bases deletions confirmed by DNA sequencing. A different sequence at codon 53-54 (C-G rather than G-C) was also found in all of the liver and tumor tissues. Our study indicates that alterations of CDKN2 gene is not a rare phenomenon in HCC, however, it might play an important role in the development of HCC and codon 81 might be a mutational hotspot of CDKN2 gene in HCC.

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.

Cholangiocarcinomas are rare but aggressive cancers, with an increasing incidence and a dismal 5-year survival rate of less than 5%. Mutations in the genes encoding isocitrate dehydrogenase, IDH1 and IDH2, have been reported in ∼70% of gliomas, ∼20% of myeloid leukemias, ∼56% of chondrosarcomas, and ∼10% of melanomas. We discovered IDH1 and IDH2 mutations in 29 of 305 (9%) intrahepatic cholangiocarcinomas. Tumors with mutations in IDH1 or IDH2 had lower 5-hydroxymethylcytosine and higher 5-methylcytosine levels, as well as increased dimethylation of histone H3K79. Mutations in IDH1 or IDH2 were independently associated with longer time to recurrence of cholangiocarcinoma in multivariate analysis (p = 0.046). We identified 2,267 genes that were significantly hypermethylated in tumors with mutations in IDH1 or IDH2. Hypermethylated CpG sites were significantly enriched in CpG shores and in the 1500 bp upstream of annotated transcription start sites, suggesting a global regulation of transcriptional potential. Gene expression profiling of 7 IDH1/2-mutant and 20 IDH1/2-wildtype tumors revealed increased expression of citric acid cycle and oxidative phosphorylation enzymes, as well as decreased expression of cytoskeleton organization genes. Integrated analyses of DNA methylation and gene expression identified interleukin-6 and several of its downstream targets as candidates for silencing by DNA methylation. Conclusions: Cholangiocarcinomas with mutations in IDH1 or IDH2 represent a molecular subclass with distinct signaling pathway alterations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1058. doi:1538-7445.AM2012-1058

Purpose: Early tumor recurrence after liver resection for hepatocellular carcinoma (HCC) was mainly due to intrahepatic tumor spreading. In this study, we set out to identify specific microRNA (miRNA) which might be used in predicting the possibility of early recurrence after HCC resection. Experimental Design: Taqman low density arrays were used to detect the miRNA expression levels in both the microdissected tumorous and adjacent non-tumorous liver tissues from 20 HCC patients. Quantitative real-time PCR (qRT-PCR) was used to verify the findings in 106 patients, and to develop a predictive assay. The identified miRNAs were further validated in an independent cohort of 112 patients. Results: Thirty seven selected miRNAs were validated in 106 HCC patients using qRT-PCR. A significant association was found between miR-29a* level in HCC tissues and early recurrence (P=0.0002). This association was further confirmed in the independent testing set of 112 patients (P=0.0154), in which miR-29a* level was significantly associated with both time to tumor recurrence (TTR) (P=0.0015) and overall survival (OS) (P=0.0079) of patients. In the univariate and multivariate analyses, miR-29a* was identified as an independent factor for TTR, particularly for those patients with early stage of HCC. The sensitivity and specificity of miR-29a* for the prediction of early HCC recurrence in an independent blinded validation set reached 74.2% and 68.2%, respectively. Conclusion: MiR-29a* might be a useful marker for prediction of early recurrence of HCC, particularly for those with early stage of HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 718. doi:1538-7445.AM2012-718

Hepatocellular carcinoma (HCC) is an aggressive solid tumor with vast heterogeneity in its clinical presentation and tumor biology. Genomic and transcriptomic analyses have revealed numerous HCC subtypes that are associated with patient outcome. Recently, we have identified a subset of aggressive HCC with stem cell-like traits and elevated levels of EpCAM and AFP (EpCAMhigh/AFPhigh). To identify key pathways contributing to this subset of HCCs, we used an integrated genomic strategy by comparing gene expression profiles of a well-defined HCC cohort consisting of 38 EpCAMhigh/AFPhigh and 40 EpCAM−/AFP− HCC cases using Affymetrix U133A array analyses. We identified a robust gene expression signature that can classify these two subgroups and is predictive of HCC prognosis. We validated this signature as an independent predictor of patient survival in two additional independent cohorts of HCC samples with mixed etiologies and ethnicity. Gene Set Enrichment Analysis revealed that genes containing YY1 transcriptional factor binding sites are most significantly enriched in the signature, suggesting that the YY1 signaling pathway may be functionally critical in regulating stem cell-like HCC. Genomic analysis using the Agilent 105A human arrayCGH platform revealed that the YY1 co-activator gene YY1AP1, but not YY1, is highly amplified in a majority of EpCAMhigh/AFPhigh HCCs (85%). YY1AP1 expression in HCC was significantly correlated with its genomic amplification and was also associated with patient prognosis. Using a lentiviral construct expressing YY1AP1 shRNA either constitutively or inducibly via tetracycline, we found that silencing of YY1AP1 completely blocked the growth of EpCAMhigh/AFPhigh HCC cell lines that contain YY1AP1 gene amplification. Furthermore, we found that YY1 transcriptionally activated YY1AP1-dependent EpCAM expression. In parallel, silencing of EpCAM via RNA interference also inhibited HCC cell growth. Our results suggest that YY1-YY1AP1 signaling is an oncogenic driver critical to the development of stem cell-like HCC through activation of EpCAM. Our results also demonstrate the feasibility and power of our strategy to utilize well-defined patient samples to uncover critical pathways linked to HCC subtypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-408. doi:1538-7445.AM2012-LB-408

In our previous study, osteopontin (OPN) was identified as one of the leading genes that promote the metastasis of hepatocellular carcinoma (HCC), and a potential therapeutic target for HCC metastasis. The serine protease thrombin can interact with OPN and modify its biologic activity. To explore the role of thrombin alone or collaborating with OPN in HCC, we investigated the thrombin level and its correlation to the prognosis of 72 HCCs with different OPN backgrounds using real-time PCR and Western blot, and evaluated the effects of OPN fragments formed by thrombin cleavage on in vitro proliferation and adhesion of HCC cells. We found that the expression level of thrombin was positively related to the metastatic potentials of HCC cell lines, and was remarkably overexpressed in HCCs compared with the adjacent nontumor tissues. In addition, HCC tissues from patients with recurrent disease displayed much higher thrombin levels, particularly in those with OPN overexpressed. In the immunohistochemical study of 230 HCCs, only in those with a higher OPN level, a significant association was found between the higher thrombin level and the overall survival (OS; P < 0.01) and time to recurrence (TTR; P < 0.0001) of patients. Assays in vitro demonstrated that thrombin could promote the proliferation and adhesion of OPN+ HCC cells. And N-terminal of OPN showed better effects on the proliferation and adhesion abilities of HCC cells than the intact protein. Thrombin treatment could activate FAK pathway of OPN+ HCC cells, and this activation was blocked by the inhibition of integrin β1. Conclusions: Thrombin plays an important role in OPN-mediated aggressive phenotype and poor prognosis of HCC, which may be through the activation of the integrin β1 -FAK signaling. So, thrombin might be a potential therapeutic target to combat HCC progression in thrombin+/OPN+ patients. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A16.

Hepatocellular carcinoma (HCC) is the most frequent malignant tumor in the liver and the third leading cause of cancer death worldwide. The high mortality of HCC is mainly caused by intra-hepatic metastasis or the high rate of recurrence after surgery. Despite potentially curative surgical resection, eligibility is scarce and patients suffer from a high recurrence rate. Recently, we developed a tumor-based 153-gene signature that is predictive of HCC portal venous metastasis and survival in 40 HCC patients (Ye et al, Nat Med, 9: 416-23, 2003). This signature was based on cDNA microarray profiling of 20 well defined HCC cases, in which ten developed tumor thrombi in the major branch of portal vein and ten had no metastasis at one year follow-up. In this study, we sought to validate this signature for its utility in predicting HCC survival using two independent cohorts. The first cohort consisted of 247 predominantly HBV+ HCC cases analyzed by the Affymetrix U133A 2.0 platform. The second cohort included 139 cases with mixed etiology analyzed by the Human Array-Ready Oligo Set (version 2) array platform. We employed a survival risk prediction algorithm with 10-fold cross validation and 1000 random permutation to test whether the 153-gene signature is associated with survival. We found that this gene signature was able to predict prognosis in both independent HCC cohorts. Importantly, the risk prediction was independent of the microarray platform and etiology. In addition, survival prediction was successful in patients with small ( Supported by NIH intramural research funds Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4818.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. Like most solid tumors, HCC is very heterogenous in its clinical presentation and genetic makeup, due to genomic instability. The poor survival of HCC patients is partly due to inefficient treatment regimens and the lack of accurate prognostic subgroup stratifications. The discovery of genomic changes in other cancer types has led to the development of novel specific therapies, underscoring the importance of understanding molecular mechanism and tumor biology to provide the best therapy for each cancer patient. To this end, we performed high-resolution array-based comparative genomic hybridization of 76 HCC clinical specimens (cohort1) to search for genetically disrupted genes associated with outcome. In agreement with previous publications, recurrent gains and losses were associated with 1q, 6p, 8q and 4q, 8p, 13q, 16, 17p, respectively. However, only deletions of a few distinct loci on 1p,4q, 8p and 9p were associated with survival (FDR0.3, p Citation Information: Cancer Res 2009;69(23 Suppl):A25.

Background & Aims Cancer progression/metastases and embryonic development share many properties including cellular plasticity, dynamic cell motility, and integral interaction with the microenvironment. We hypothesized that the heterogeneous nature of hepatocellular carcinoma (HCC), in part, may be owing to the presence of hepatic cancer cells with stem/progenitor features. Methods Gene expression profiling and immunohistochemistry analyses were used to analyze 235 tumor specimens derived from 2 recently identified HCC subtypes (EpCAM + α-fetoprotein [AFP + ] HCC and EpCAM − AFP − HCC). These subtypes differed in their expression of AFP, a molecule produced in the developing embryo, and EpCAM, a cell surface hepatic stem cell marker. Fluorescence-activated cell sorting was used to isolate EpCAM + HCC cells, which were tested for hepatic stem/progenitor cell properties. Results Gene expression and pathway analyses revealed that the EpCAM + AFP + HCC subtype had features of hepatic stem/progenitor cells. Indeed, the fluorescence-activated cell sorting–isolated EpCAM + HCC cells displayed hepatic cancer stem cell–like traits including the abilities to self-renew and differentiate. Moreover, these cells were capable of initiating highly invasive HCC in nonobese diabetic, severe combined immunodeficient mice. Activation of Wnt/β-catenin signaling enriched the EpCAM + cell population, whereas RNA interference-based blockage of EpCAM, a Wnt/β-catenin signaling target, attenuated the activities of these cells. Conclusions Taken together, our results suggest that HCC growth and invasiveness is dictated by a subset of EpCAM + cells, opening a new avenue for HCC cancer cell eradication by targeting Wnt/β-catenin signaling components such as EpCAM.

AIM: To quantify the circulating DNA in plasma from patients with hepatocellular carcinoma (HCC) and to evaluate its prognostic value. METHODS: Blood samples were collected from 79 patients with HCC before operation, 20 patients with liver cirrhosis, and 20 healthy volunteers. Circulating DNA was extracted from plasma and quantified. The association between circulating DNA level and prognosis of HCC patients was evaluated. RESULTS: Compared with the healthy volunteers (17.6 ± 9.5 ng/mL), a significant higher circulating DNA level was found in the patients with HCC (47.1 ± 43.7 ng/mL, P = 0.000) or with liver cirrhosis (30.0 ± 13.3 ng/mL, P = 0.002). The circulating DNA level was closely associated with tumor size (P = 0.008) and TNM stage (P = 0.040), negatively associated with the 3-year disease-free survival (DFS) (P = 0.017) and overall survival (OS) (P = 0.001). CONCLUSION: Large or invasive tumor may release more circulating DNA, and higher level of circulating DNA may be associated with poor prognosis of HCC patients.

AIM: To evaluate the effect of postoperative adjuvant transcatheter arterial chemoembolization (TACE) on the prognosis of hepatocellular carcinoma (HCC) patients with or without risk factors for the residual tumor. METHODS: From January 1995 to December 1998, 549 consecutive HCC patients undergoing surgical resection were included in this research. There were 185 patients who underwent surgical resection with adjuvant TACE and 364 patients who underwent surgical resection only. Tumors with a diameter more than 5 cm, multiple nodules, and vascular invasion were defined as risk factors for residual tumor and used for patient stratification. Kaplan-Meier method was used to analyze survival curve and Cox proportional hazard model was used to evaluate the prognostic significance of adjuvant TACE. RESULTS: In the patients without any risk factors for the residual tumor, the 1-, 3-, 5-year survival rates were 93.48%, 75.85%, 62.39% in the control group and 97.39%, 70.37%, 50.85% in the adjuvant TACE group, respectively. There was no significant difference in the survival between two groups (P = 0.3956). However, in the patients with risk factors for residual tumor, postoperative adjuvant TACE significantly prolonged the patients’ survival. There was a statistically significant difference in survival between two groups (P = 0.0216). The 1-, 3-, 5-year survival rates were 69.95%, 49.86%, 37.40% in the control group and 89.67%, 61.28%, 44.36% in the adjuvant TACE group, respectively. Cox proportional hazard model showed that tumor diameter and cirrhosis, but not the adjuvant TACE, were the significantly independent prognostic factors in the patients without risk factors for residual tumor. However, in the patients with risk factors for residual tumor adjuvant TACE, and also tumor diameter, AFP level, vascular invasion, were the significantly independent factors associated with the decreasing risk for patients’ death from HCC. CONCLUSION: Postoperative adjuvant TACE can prolong the survival of patients with risk factors for residual tumor, but can not prolong the survival of patients without risk factors for residual tumor.

The prognosis of hepatocellular carcinoma (HCC) still remains dismal, although many advances in its clinical study have been made. It is important for tumor control to identify the factors that predispose patients to death. With new discoveries in cancer biology, the pathological and biological prognostic factors of HCC have been studied quite extensively. Analyzing molecular markers (biomarkers) with prognostic significance is a complementary method. A large number of molecular factors have been shown to associate with the invasiveness of HCC, and have potential prognostic significance. One important aspect is the analysis of molecular markers for the cellular malignancy phenotype. These include alterations in DNA ploidy, cellular proliferation markers (PCNA, Ki-67, Mcm2, MIB1, MIA, and CSE1L/CAS protein), nuclear morphology, the p53 gene and its related molecule MD M2, other cell cycle regulators (cyclin A, cyclin D, cyclin E, cdc2, p27, p73), oncogenes and their receptors (such as ras, c-myc, c-fms, HGF, c-met, and erb-B receptor family members), apoptosis related factors (Fas and FasL), as well as telomerase activity. Another important aspect is the analysis of molecular markers involved in the process of cancer invasion and metastasis. Adhesion molecules (E-cadherin, catenins, serum intercellular adhesion molecule-1, CD44 variants), proteinases involved in the degradation of extracellular matrix (MMP-2, MMP-9, uPA, uPAR, PAI), as well as other molecules have been regarded as biomarkers for the malignant phenotype of HCC, and are related to prognosis and therapeutic outcomes. Tumor angiogenesis is critical to both the growth and metastasis of cancers including HCC, and has drawn much attention in recent years. Many angiogenesis-related markers, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PD-ECGF), thrombospondin (TSP), angiogenin, pleiotrophin, and endostatin (ES) levels, as well as intratumor microvessel density (MVD) have been evaluated and found to be of prognostic significance. Body fluid (particularly blood and urinary) testing for biomarkers is easily accessible and useful in clinical patients. The prognostic significance of circulating DNA in plasma or serum, and its genetic alterations in HCC are other important trends. More attention should be paid to these two areas in future. As the progress of the human genome project advances, so does a clearer understanding of tumor biology, and more and more new prognostic markers with high sensitivity and specificity will be found and used in clinical assays. However, the combination of some items, i.e., the pathological features and some biomarkers mentioned above, seems to be more practical for now.

The prognosis of patients with HCC still remains dismal. The life expectancy of HCC patients is hard to predict because of the high possibility of postoperative recurrence. Many factors, such as patient's general conditions, macroscopic tumor morphology, as well as tumor histopathology features, have been proven of prognostic significance. Female HCC patient often has a better prognosis than male patient, which might be due to the receptor of sex hormones. Younger patients often have tumors with higher invasiveness and metastatic potentials, and their survival and prognosis are worse than the older ones. Co-existing hepatitis status and hepatic functional reserve have been confirmed as risk factors for recurrence. Serum alpha-fetoprotein (AFP) is useful not only for diagnosis, but also as a prognostic indicator for HCC patients. AFP mRNA has been proposed as a predictive marker of HCC cells disseminated into the circulation and for metastatic recurrence. Many pathologic features, such as tumor size, number, capsule state, cell differentiation, venous invasion, intrahepatic spreading, and advanced pTNM stage, are the best-established risk factors for recurrence and important aspects affecting the prognosis of patients with HCC. Marked inflammatory cell infiltration in the tumor could predict a better prognosis. Clinical stage is still the most important factor influencing on the prognosis. Extratumor spreading and lymph nodal metastasis are independent predictors for poor outcome. Some new predictive systems have recently been proposed. Different strategies of treatment might have significant different effects on the patients' prognosis. To date, surgical resection is still the only potentially curative treatment for HCC, including localized postoperative recurrences. Extent of resection, blood transfusion, occlusion of porta hepatis, and blood loss affect the survival and prognosis of HCC patients. Regional therapies provide alternative ways to improve the prognosis of HCC patients who have no opportunity to receive surgical treatment or postoperative recurrence. The combination of these treatment modalities is hopeful to further improve the prognosis. The efficacies of neoadjuvant (preoperative) or adjuvant (postoperative) chemotherapy or chemoembolization in preventing recurrence and on the HCC prognosis still remain great controversy, and deserve further evaluation. Biotherapy, including IFN-alpha therapy, will play more important role in preventing recurrence and metastasis of HCC after operation.

Metastatic human HCC model is needed for the studies on mechanism and interven tion of metastatic recurrence. By using orthotopic implantation of histologically intact tissues of 30 surgical specimens, a patient-like metastatic model of hu man HCC in nude mice (LCI-D20) and a low metastatic model of human HCC in nude mice (LCI-D35) have been established. All mice with transplanted LCI-D20 tumors exhibited extremely high metastatic ability including spontaneous metasta sis to liver, lungs, lymph nodes and peritoneal seeding. Remarkable difference was also found in expression of some of the invasiveness related genes and growth factors between the LCI-D20 and LCI-D35 tumors. PAI-1 increased gradually following tumor progression in LCI-D20 model, and correlated with tumor size and AFP level. Phasic expression of tissue intercellular adhesio nmolecule-1 in this model was also observed. Using corneal micropocket model, it was demonstrated that the vascular response induced by LCI-D20 tumor was stronger than that induced by LCI-D35 tumor. Similar report on metastatic human HCC model in nude mice and human HCC cell line with metastatic potential was rarely found in the literature. This LCI-D20 model has been widely used for the studies on intervention of metastasis, including anti-angiogenesis, antisense approach, metallopro teinase inhibitor, differentiation inducer, etc. It is concluded that the establ ishment of metastatic human HCC model in nude mice and human HCC cell line with metastatic potential will provide important models for the in vivo and in vitro study of HCC invasiveness, angiogenesis as well as intervention of HCC recurrence.