Translational medicine in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a highly complex disease that is generally resistant to commonly used chemotherapy and radiotherapy. Consequently, there is an urgent need for the development of new treatment strategies for this devastating disease. In the past decade, tremendous progress has been achieved in the molecular stratification of HCCs for diagnosis, prognosis, and therapeutic decision-making. To date, the molecular classification of HCCs has been carried out through transcriptomic, genetic and epigenetic profiling of tumors. Such research has led to identification of several potential molecular targets in HCC, and subsequently, development of novel systemic agents for the treatment of HCC has begun in earnest. In this article, we review the current knowledge of the molecular pathogenesis of HCC and outline potential areas for application of this knowledge in a clinical setting. As a typical virus and inflammation-associated cancer, both host immune response and tumor microenvironment have crucial roles in HCC pathogenesis. In addition, we examine the potential of immunotherapy and strategies targeting various components of the tumor microenvironment, as well as novel molecular and cellular targets in HCC such as cancer stem cells.

Keywords

hepatocellular carcinoma / molecular classification / molecular targeted therapies / tumor microenvironment / immunotherapy

Introduction

Hepatocellular carcinoma (HCC) ranks among the most common cancers worldwide and is the third leading cause of cancer death, accounting for more than half a million deaths annually [1]. Although prevalence remains highest in Eastern Asia and Africa, the incidence of HCC has steadily increased in the western world, as well as in Japan over the last decade [2]. HCC represents the most relevant paradigm of virus and inflammation-associated cancers, suggesting that primary and secondary prevention of HCC is possible. Meanwhile, the majority (>80%) of HCC patients suffer complications such as chronic hepatitis or cirrhosis. Such complications influence disease progression and severely impair patient prognosis, therapeutic options, and the design of clinical trials [3]. Despite its enormous clinical relevance, knowledge regarding the molecular pathogenesis of HCC lags behind other types of cancers, such as breast and colon cancer, where multiple systemic treatment options have evolved for previously untreatable metastatic tumors. Recently, this picture has begun to change for HCC, as improved options for clinical trials and novel therapeutics are emerging owing to the identification of critical molecular subclasses of HCC with different prognostic implications, and the discovery of key genetic or epigenetic drivers of specific subclasses that enable development of more personalized treatments.

Molecular classification and prognostication of HCC

The aim of cancer classification is to better predict patient prognosis, guide the selection of an appropriate treatment for the best candidates, and aid researchers in the design of clinical trials with comparable clinical criteria. In HCC, 15%-20% of early stage tumors present with a dismal outcome, leading to neoplastic dissemination and short-term survival. The identification of biomarkers that help detect subgroups of patients with a dismal prognosis, even for those in an early disease stage, will translate into better therapeutic strategies and allocation of resources. Currently, the prediction of patient prognosis in HCC relies exclusively on clinical parameters, and molecular data are not utilized to guide therapeutic decision-making. This represents a critical bottleneck for the improvement of patient outcome.

The first attempts to classify HCC were based on oncogene or tumor suppressor activity. The most common mutation in HCC is found in the tumor suppressor TP53, observed in up to 50% of HCC cases [4]. The second most common mutation of a specific gene is activation of the oncogene β-catenin, found in 20%-40% of HCC [5]. Other low or moderate frequency mutations have been described for various oncogenes such as KRAS, PIK3CA, MET, and CSF-1R and tumor suppressors including CDKN2A, AXIN1/2, RB1, PTEN, IGF2R, and SMAD2/4 [6,7]. Undoubtedly, the spectrum of mutations will be expanded by the use of new-generation high-throughput sequencing technology in the near future [8]. Through exonic sequencing of ten hepatitis C virus (HCV)-associated HCCs and subsequent validation, novel inactivating mutations of the gene, ARID2, were identified in four major subtypes of HCC (HCV-related, hepatitis B (HBV)-related, alcohol-related HCC and HCC with subtle etiology), suggesting that ARID2 is a tumor suppressor that is commonly mutated in HCC, irrespective of disease etiology [9].

Although gene mutations are clearly linked to HCC biologic behavior, they are inadequate for accurate classification of HCC. With the advent of high throughput genomic, transcriptomic and epigenetic techniques, it is now possible to inspect the whole genome for different functional aspects. (i) At the transcriptional level, several molecular subgroups of tumors have been identified, underlining the broad diversity of HCC in humans. These molecular subgroups were obtained using the expression patterns of genes involved in cell proliferation and cell cycling [10] or other hallmark signaling of tumors [11], and genes typically expressed in hepatic progenitor cells [12]. Notably, expression patterns of genes involved in the tumor-stroma interaction were also utilized to predict patient prognosis, highlighting the importance of stromal biology [13] in HCC tumor progression. (ii) MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression, could also act as oncogenes or tumor suppressors [14]. A relationship between miRNA deregulation and the diagnosis and phenotype of HCC has been reported [15,16]. Importantly, a diagnostic miRNA panel has been derived using patient plasma and its future clinical use for early diagnosis of HCC can be envisioned [16]. In addition, low expression of miR-26a [17,18], miR-122 [19,20] and miR-199a/b-3p [21], as well as high expression of miR-221 [22,23] were found to be significantly associated with HCC recurrence. Despite observed differences in the clinical features of the tumors and patient risk factors, as well as variations in techniques and strategies used to normalize tumor profiling data, recurrent alterations in the expression of several miRNAs have been found in a number of studies. This suggests that HCCs may have a distinct miRNA expression fingerprint. (iii) Methylation changes may occur early in the process of hepatocarcinogenesis, and HBV, HCV, and alcohol-specific promoter methylation patterns have been described, suggesting that etiology-dependent methylation occurs in the early stages of hepatocarcinogenesis [24]. The methylation index for 105 tumor suppressor genes was shown to have a significant inverse correlation with tumor recurrence after hepatectomy, as well as cancer-free and overall survival [25]. Despite the achievement of great progress in the field, several challenges have limited the successful translation of such findings for clinical applications. These include the lack of consistency in study results, as well as the weak associations often found with clinical phenotype. Varying etiologies, inadequate sample sizes, and differences in microarray techniques utilized, as well as differences in mathematical methods and experimental models used, significantly contribute to the existing limitations and should be taken into account for future studies.

In HCC, two types of recurrence can arise from a distinct biologic background: true metastasis as dissemination of primary tumor cells (usually within 2 years after curative treatment) and de novo metachronic tumors occurring in a diseased liver [26]. The proportion of each type of recurrence varies greatly across tumor stages. Importantly, gene expression profiling of HCCs, a 19-miRNA signature and a methylation profile, all derived from the adjacent non-tumoral liver tissue instead of HCC tissue, were used to accurately identify patients with poor prognosis [27-29]. The observed accuracy was probably due to the ability to identify the risk of developing de novo tumors, progression of liver dysfunction, and microenvironmental-favoring conditions for intrahepatic metastasis. Thus, these signatures can be used to identify the risk of progression of cirrhosis, and might be relevant guides for chemopreventive studies. Cirrhosis, a life-threatening condition, is present in more than 80% of patients with HCC, making prognosis prediction a major challenge. As such, any staging system for HCC based on molecular data should incorporate information related to tumor aggressiveness, hepatic dysfunction, and risk of de novo HCC development [30]. Hence, such a prognostic model should consider genetic, transcriptomic, and epigenetic data coded in the tumor itself, as well as adjacent non-tumoral cirrhotic tissue. From early to intermediate and advanced stages, genomic data derived from the tumor will be increasingly informative when compared with adjacent non-tumor, because, as cancer progresses, tumor dissemination will govern patient survival. The scientific challenge is to identify the genomic vector that determines patient prognosis at each stage of the disease. A further application of such data would be for the development of molecular markers used in a clinical setting. PCR-based techniques for detecting genes of interest appear to be more robust and potentially clinical usable, but are only feasible when large gene panels are refined to a limited number of genes.

Targeted molecular therapies in HCC

Less than 30% of newly diagnosed patients are eligible for curative therapies such as resection, liver transplantation, or local ablation. Furthermore, HCC is highly resistant to conventional systemic therapies and no level I studies have so far shown survival benefits of conventional chemotherapy in HCC patients. Targeted molecular therapies for the treatment of cancer including HCC are considered a promising approach, as demonstrated by the breakthrough results of the multi-kinase inhibitor, sorafenib. Sorafenib is the first compound found to significantly improve the survival of patients with advanced HCC, with a mean survival benefit of 3 months [31]. This multi-kinase inhibitor is active against various receptor tyrosine and serine threonine kinases, including Raf-1, vascular endothelial growth factor receptor 2 (VEGFR2), B-Raf, platelet-derived growth factor (PDGFR) and c-Kit. In addition to sorafenib, many novel compounds are currently under preclinical and clinical evaluation for HCC (www.clinicaltrials.gov) (Table 1). Treatment agents are generally classified according to their main targets: angiogenic factors, growth factors and their receptors, and intracellular targets.

The overexpression of angiogenic factors such as VEGF, PDGF, fibroblast growth factor 2 (FGF-2), or angiopoietin are early and frequent events in hepatocarcinogenesis [33]. Blocking angiogenesis through the targeting of microvascular endothelial cells or protein activators of angiogenesis is a well-accepted strategy for preventing HCC tumor progression. The humanized monoclonal antibody, Bevacizumab (anti-VEGF), is approved for the treatment of breast cancer and colorectal liver metastases. Bevacizumab showed modest anti-tumoral activity in a phase I/II trial of 33 selected HCC patients, as well as in a study that combined treatment with the chemotherapy agents, gemcitabine and oxaliplatin [34]. FGF2 has been shown to augment VEGF-mediated HCC development and angiogenesis in a synergistic manner, and may play a role in “escape mechanisms” implicated in VEGF/VEGFR targeting [35]. Interestingly, brivanib, a small molecule inhibiting FGF and VEGFR, is currently under investigation in phase III trials after showing promising results as a second line agent after failure of patients to respond to sorafenib treatment. In addition, TSU-68 is a small molecule inhibitor of the angiogenic receptor tyrosine kinases Flk-1/KDR, PDGFRβ, and FGFR1, currently in phase I/II clinical trials for HCC.

Peptide growth factors and their receptors such as epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (HGFR)/c-Met, and insulin-like growth factor receptor (IGFR) are also well-defined pathways of potential interest in HCC. Single-agent studies with EGFR-directed therapies in HCC have been disappointing: only erlotinib showed anti-tumoral activity in experimental models and clinical studies [36], whereas clinical trials involving other drugs such as gefitinib, laptinib and vandetanib either reported no activity or required further studies [37,38]. Preclinical data suggest that only a more “epithelial” subtype of HCC, as defined by the expression of E-cadherin (versus the presence of vimentin and absence of E-cadherin in more “mesenchymal” subtypes), may be dependent on EGFR signaling [39], suggesting a specific target population may need to be identified for EGFR agents to be clinically effective. The IGF signaling axis is frequently activated in HCC and several IGF-1R tyrosine kinase inhibitors have been tested preclinically for HCC. Meanwhile, BIIB022 and AVE 1642 are two novel drugs targeting IGF signaling, which are currently in phase I/II studies for use as monotherapies or in combination with sorafenib and erlotinib, respectively. The clinical efficacy of targeting HGFR/c-Met has yet to be determined, and several studies with receptor and ligand-specific antibodies, as well as tyrosine kinase inhibitors are ongoing or in development [40].

Lastly, the biologic effects of receptor tyrosine kinase activation are mediated by a complex cascade of intercellular signaling molecules that are potential targets for therapy. For example, mTOR-inhibitors are of increasing interest for the treatment of HCC, as activation of the PI3K/Akt/mTOR pathway was reported for up to 50% of HCC [41]. An mTOR-inhibitor, everolimus, demonstrated antitumor activity in experimental HCC models [42], which led to its testing in phase II and III trials [43]. The mTOR inhibitor rapamycin, which has been shown to prevent organ rejection, is also being tested in patients with HCC who received liver transplantation. Interestingly, patients treated with rapamycin were found to have a lower risk of recurrence of HCC [44,45]. Other key signal transduction pathways implicated in the pathogenesis of HCC that have therapeutic potential are the Ras/Raf/MAPK pathway, Wnt-β-Catenin pathway, Hedgehog signaling, JAK-STAT pathway, and apoptotic pathways [46]. Deregulated apoptosis and autophagy synergistically promote HCC invasion and metastasis, and represent valuable molecular targets for intervention to reduce metastasis and recurrence [47-49]. Phase I trials targeting the apoptosis inhibitors XIAP and BCL2 are ongoing in HCC.

A further approach in the treatment of HCC is targeting the epigenetic changes that inactivate important tumor suppressor genes or pathway antagonists. The potential use of antagomirs and reconstitutive miRNA compounds in the treatment of cancer is very promising. There is emerging evidence for the use of combination therapies targeting various molecules for adjuvant therapy. Unraveling the complex interactions between diverse biologic levels and dissecting the different signaling pathways activated during hepatocarcinogenesis will certainly lead to the development of more effective combination strategies for treatment of HCC. Nonetheless, the ultimate biomarker to predict treatment responses has not been identified. It should be emphasized that pharmacogenomic analyses of patients should be carried out during clinical trials to test molecular therapies in order to determine the optimal combination of available targeted therapies and to better understand the mechanisms of drug resistance.

The tumor microenvironment as a therapeutic target in HCC

The microenvironment of HCC is composed of cellular components such as hepatic stellate cells, fibroblasts, immune, and endothelial cells, as well as various non-cellular components, including extracellular matrix (ECM) proteins, proteolytic enzymes, growth factors and inflammatory cytokines [50]. The cross-talk between HCC cells and the surrounding microenvironment is a key modulator of the processes of hepatocarcinogenesis, tumor invasion and metastasis. Previously, HCC treatments were focused on targeting tumor cells. However, biologic agents that target components of the tumor microenvironment may provide an interesting alternative to traditional tumor cell-directed therapy. In this regard, the multikinase inhibitor sorafenib represents a typical reagent that simultaneously blocks tumor angiogenesis while decreasing HCC cell viability, and inducing apoptosis.

In particular, targeted treatment aimed at inhibiting TGF-β signaling appears to be promising, as high expression of TGF-β is a key mediator of liver fibrosis, HCC progression, and epithelial-mesenchymal transition (EMT), in addition to being a poor prognostic indicator of HCC [51-53]. The TGF-β receptor 1 kinase inhibitor, LY2109761, deactivates Smad-2 protein, decreasing the migration and vascular invasion of HCC cells and upregulating E-cadherin expression in HCC cell membranes, which in turn mediates cell adhesion. LY2109761 was shown to inhibit tumor-specific neoangiogenesis by blocking paracrine cross-talk between HCC and endothelial cells, via Smad-2 dependent inhibition of VEGF production. The observed efficacy of LY2109761 was surprisingly superior to bevacizumab, which specifically targets VEGF [51,52]. In addition, LY2109761 was also shown to inhibit the cross-talk between HCC cells and cancer-associated fibroblasts, through the downregulation of connective tissue growth factor, thus inhibiting tumor progression [51,52]. More recently, the interaction of CD151 in HCC cells and its ligand laminin-5 in ECM was demonstrated to promote the spread of HCC by inducing EMT and neoangiogenesis. These findings suggested that CD151 was a promising therapeutic target that functioned by dampening cross-talk between HCC and ECM components [54-56]. In addition, mesenchymal stem cells that overexpressed the pigment epithelium-derived factor were able to inhibit the growth of HCC in nude mice, which may make it a useful therapeutic tool in the control of HCC [57]. Nonetheless, phase I clinical trials targeting TGF-β signaling, CD151 or other components of the tumor microenvironment have not yet been performed.

The basic rationale for targeting tumor-stromal interaction is to suppress the effect of surrounding tissues or cell types on hepatocarcinogenesis, tumor progression, invasion, and metastasis, while minimizing systemic toxicity by delivering drug effects specifically to tumors and their microenvironment. It should be noted that each component of the tumor microenvironment shares some functional redundancies. Therefore, targeting one molecular component of the tumor microenvironment does not necessarily suppress HCC progression. Of particular importance, the tumor microenvironment of HCC consists of the intratumoral microenvironment and the peritumoral liver microenvironment, both of which play a crucial role in HCC invasion and metastasis [58-61].

Potential of immunotherapy for HCC

Immunotherapy aims to provide a more efficient and selective targeting of tumor cells, by inducing or boosting the existing tumor-specific immune response. The rationale for immunotherapy for HCC is based on the finding that patients with tumors containing infiltrating, presumably tumor-specific cytotoxic cells and memory T cells, had a reduced risk of tumor recurrence following hepatectomy and liver transplantation [61,62]. Moreover, anti-CD3 and interleukin (IL)-2-stimulated autologous T lymphocytes significantly improved post-surgical recurrence-free survival when infused in HCC patients [63]. These data imply a central role of T cells in modulating tumor progression and provide strong justification for T cell immunotherapy. As such, a number of immunotherapeutic options were evaluated in HCC patients (Table 2) and phase II trials targeting PD-1 or B7-CD28 are now ongoing in HCC patients.

Tumor-associated antigens (TAAs) are required for the successful development of T cell-based immunotherapies. To date, six HCC-specific TAAs targeted by T cells have been identified, including α-fetoprotein, glypican-3, NY-ESO-1, SSX-2, melanoma antigen gene-A, and telomerase reverse transcriptase [91]. The frequency of these TAA-specific T cell responses is low in HCC patients and in vitro stimulation is often required for their detection. Despite the development of multiple approaches for the use of T cell-based HCC immunotherapies so far, the tumor-specific T cell responses achieved were often not robust enough to induce clinical responses. The observed modest clinical efficacy may be explained by the strong tolerogenic environment at the tumor site and the existence of various tumor suppressive factors, including regulatory T cells, tumor associated macrophages and neutrophils, myeloid-derived suppressor cells, and tumor-derived inhibitory factors (Fig. 1) [58,60,61,92,93]. Clearly, treatment strategies combining the blockade of immunoregulatory cell types such as regulatory T cells, myeloid-derived suppressor cells and inhibitory receptors, along with vaccine-induced activation of TAA-specific T cells may be necessary to achieve the most effective therapeutic antitumor activity in HCC. Importantly, a combination of the above strategies with conventional HCC treatments, such as transarterial embolization or local tumor ablation may increase HCC immunogenicity and unmask TAA-specific T cell responses.

A role for liver cancer stem cells

The hierarchical model of cancer origin is based on the assumption that tumor heterogeneity originates from a small population of cancer stem cells (CSCs) that share multiple characteristics of tissue stem cells. The failure of existing treatments for cancer has initiated the search for new methods that effectively target CSCs. However, CSCs are difficult to treat using conventional methods because of their chemoresistant and radioresistant properties, as well as their ability to stimulate angiogenesis. Moreover, these properties of CSCs contribute to tumor recurrence and treatment resistance in advanced cases of cancer, through increased levels of BCL2 family proteins or activation of ABC transporters [94].

In HCC, efforts have been focused on identifying markers exclusive to liver CSCs and toward developing targeted therapies. The Hoechst 33342 dye has been used to isolate liver CSC-like side population cells [95]. Numerous studies have been published on various markers for putative human liver CSCs: specifically CD133, CD90, epithelial cell adhesion molecule (EpCAM) and CD13 [96-99]. Specific targeting of liver CSC in pre-clinical studies, for instance, by targeting EpCAM ⁺ cells by RNAi-mediated inhibition or forced differentiation using the cytokine, oncostatin M in combination with conventional chemotherapy, showed increased apoptosis and decreased cell proliferation, indicating the potential of CSC for therapeutic modalities in liver cancer [97,100]. However, the use of liver CSC markers is still controversial, because none of these markers is exclusively expressed by liver CSCs in HCC. Further study of the properties of these cells may lead to modalities of therapy for HCC which specifically target liver CSCs. In addition, targeting pathways that lead to the self-renewal and proliferating properties of cancer stem cells such as the Hedgehog, Wnt, Notch, and IL-6 pathways, use of differentiation therapy such as retinoic acid, and targeting the microenvironment of the stem cell niche could all provide powerful new therapeutic approaches to treat this typically lethal and cancer [101,102].

Future perspectives

Due to efficient surveillance of patients, improved diagnostic assessment, as well as increased efforts to develop novel therapeutic strategies, liver cancer hasevolved from a universally deadly disease to a treatable one. Large scale and systematic application of novel technologies such as next-generation sequencing, as well as the study of HCC on all molecular levels including the epigenome, genome, transcriptome, and proteome are urgently needed to decode the biology of HCC and improve the limited prognosis. Anti-angiogenic strategies are likely to continue to be important in the treatment of HCC. Hence, an understanding of mechanisms of resistance to VEGF therapies will pave the way for effective treatment regimens. Further unraveling the tight and complex interactions between the diverse biologic levels and dissecting the different signaling pathways activated during hepatocarcinogenesis will certainly lead to a greater insight into HCC development and in turn, novel strategies for the diagnosis and treatment of HCC. With regards to the use of immunotherapy for HCC, the repertoire of HLA class I and class II presented TAA-specific T cell epitopes first needs to be expanded, and the mechanisms of T cell failure require further investigation before immunotherapy can successfully be utilized to treat HCC in the clinical setting. Additionally, the study of tumor-host interactions, and in particular, the tumor microenvironment, an evolving interdisciplinary field, should be considered when developing future HCC treatments. On the other hand, successful clinical development of new agents will require careful patient selection based on both the stage of underlying liver disease and tumor burden. Analyses that define the molecular heterogeneity of HCC will eventually lead to the identification of clinically useful biomarkers that are both prognostic and predictive of response to novel therapeutics.

References

[1] Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011; 61(2): 69-90

[2] Sherman M. Hepatocellular carcinoma: epidemiology, surveillance, and diagnosis. Semin Liver Dis 2010; 30(1): 3-16

[3] Bruix J, Sherman M. Management of hepatocellular carcinoma: an update. Hepatology 2011; 53(3): 1020-1022

[4] Minouchi K, Kaneko S, Kobayashi K. Mutation of p53 gene in regenerative nodules in cirrhotic liver. J Hepatol 2002; 37(2): 231-239

[5] Laurent-Puig P, Zucman-Rossi J. Genetics of hepatocellular tumors. Oncogene 2006; 25(27): 3778-3786

[6] van Malenstein H, van Pelt J, Verslype C. Molecular classification of hepatocellular carcinoma anno 2011. Eur J Cancer 2011; 47(12): 1789-1797

[7] Zucman-Rossi J. Molecular classification of hepatocellular carcinoma. Dig Liver Dis 2010; 42(Suppl 3): S235-S241

[8] Maher B. Exome sequencing takes centre stage in cancer profiling. Nature 2009; 459(7244): 146-147

[9] Li M, Zhao H, Zhang X, Wood LD, Anders RA, Choti MA, Pawlik TM, Daniel HD, Kannangai R, Offerhaus GJ, Velculescu VE, Wang L, Zhou S, Vogelstein B, Hruban RH, Papadopoulos N, Cai J, Torbenson MS, Kinzler KW. Inactivating mutations of the chromatin remodeling gene ARID2 in hepatocellular carcinoma. Nat Genet 2011; 43(9): 828-829

[10] Chiang DY, Villanueva A, Hoshida Y, Peix J, Newell P, Minguez B, LeBlanc AC, Donovan DJ, Thung SN, Solé M, Tovar V, Alsinet C, Ramos AH, Barretina J, Roayaie S, Schwartz M, Waxman S, Bruix J, Mazzaferro V, Ligon AH, Najfeld V, Friedman SL, Sellers WR, Meyerson M, Llovet JM. Focal gains of VEGFA and molecular classification of hepatocellular carcinoma. Cancer Res 2008; 68(16): 6779-6788

[11] Ye QH, Qin LX, Forgues M, He P, Kim JW, Peng AC, Simon R, Li Y, Robles AI, Chen Y, Ma ZC, Wu ZQ, Ye SL, Liu YK, Tang ZY, Wang XW. Predicting hepatitis B virus-positive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning. Nat Med 2003; 9(4): 416-423

[12] Lee JS, Heo J, Libbrecht L, Chu IS, Kaposi-Novak P, Calvisi DF, Mikaelyan A, Roberts LR, Demetris AJ, Sun Z, Nevens F, Roskams T, Thorgeirsson SS. A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells. Nat Med 2006; 12(4): 410-416

[13] Gao Q, Wang XY, Qiu SJ, Zhou J, Shi YH, Zhang BH, Fan J. Tumor stroma reaction-related gene signature predicts clinical outcome in human hepatocellular carcinoma. Cancer Sci 2011; 102(8): 1522-1531

[14] Esquela-Kerscher A, Slack FJ. Oncomirs—microRNAs with a role in cancer. Nat Rev Cancer 2006; 6(4): 259-269

[15] Toffanin S, Hoshida Y, Lachenmayer A, Villanueva A, Cabellos L, Minguez B, Savic R, Ward SC, Thung S, Chiang DY, Alsinet C, Tovar V, Roayaie S, Schwartz M, Bruix J, Waxman S, Friedman SL, Golub T, Mazzaferro V, Llovet JMMicroRNA-based classification of hepatocellular carcinoma and oncogenic role of miR-517a. Gastroenterology 2011;140(5):1618-1628. e16

[16] Zhou J, Yu L, Gao X, Hu J, Wang J, Dai Z, Wang JF, Zhang Z, Lu S, Huang X, Wang Z, Qiu S, Wang X, Yang G, Sun H, Tang Z, Wu Y, Zhu H, Fan J A plasma microRNA panel to diagnose hepatitis B virus-related hepatocellular carcinoma. J Clin Oncol 2011;29(36):4781-4788

[17] Budhu A, Jia HL, Forgues M, Liu CG, Goldstein D, Lam A, Zanetti KA, Ye QH, Qin LX, Croce CM, Tang ZY, Wang XW. Identification of metastasis-related microRNAs in hepatocellular carcinoma. Hepatology 2008; 47(3): 897-907

[18] Ji J, Shi J, Budhu A, Yu Z, Forgues M, Roessler S, Ambs S, Chen Y, Meltzer PS, Croce CM, Qin LX, Man K, Lo CM, Lee J, Ng IO, Fan J, Tang ZY, Sun HC, Wang XW. MicroRNA expression, survival, and response to interferon in liver cancer. N Engl J Med 2009; 361(15): 1437-1447

[19] Tsai WC, Hsu PW, Lai TC, Chau GY, Lin CW, Chen CM, Lin CD, Liao YL, Wang JL, Chau YP, Hsu MT, Hsiao M, Huang HD, Tsou AP. MicroRNA-122, a tumor suppressor microRNA that regulates intrahepatic metastasis of hepatocellular carcinoma. Hepatology 2009; 49(5): 1571-1582

[20] Coulouarn C, Factor VM, Andersen JB, Durkin ME, Thorgeirsson SS. Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties. Oncogene 2009; 28(40): 3526-3536

[21] Hou J, Lin L, Zhou W, Wang Z, Ding G, Dong Q, Qin L, Wu X, Zheng Y, Yang Y, Tian W, Zhang Q, Wang C, Zhang Q, Zhuang SM, Zheng L, Liang A, Tao W, Cao X. Identification of miRNomes in human liver and hepatocellular carcinoma reveals miR-199a/b-3p as therapeutic target for hepatocellular carcinoma. Cancer Cell 2011; 19(2): 232-243

[22] Pineau P, Volinia S, McJunkin K, Marchio A, Battiston C, Terris B, Mazzaferro V, Lowe SW, Croce CM, Dejean A. miR-221 overexpression contributes to liver tumorigenesis. Proc Natl Acad Sci USA 2010; 107(1): 264-269

[23] Li J, Wang Y, Yu W, Chen J, Luo J. Expression of serum miR-221 in human hepatocellular carcinoma and its prognostic significance. Biochem Biophys Res Commun 2011; 406(1): 70-73

[24] Arai E, Ushijima S, Gotoh M, Ojima H, Kosuge T, Hosoda F, Shibata T, Kondo T, Yokoi S, Imoto I, Inazawa J, Hirohashi S, Kanai Y. Genome-wide DNA methylation profiles in liver tissue at the precancerous stage and in hepatocellular carcinoma. Int J Cancer 2009; 125(12): 2854-2862

[25] Calvisi DF, Ladu S, Gorden A, Farina M, Lee JS, Conner EA, Schroeder I, Factor VM, Thorgeirsson SS. Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma. J Clin Invest 2007; 117(9): 2713-2722

[26] Sherman M. Recurrence of hepatocellular carcinoma. N Engl J Med 2008; 359(19): 2045-2047

[27] Hoshida Y, Villanueva A, Kobayashi M, Peix J, Chiang DY, Camargo A, Gupta S, Moore J, Wrobel MJ, Lerner J, Reich M, Chan JA, Glickman JN, Ikeda K, Hashimoto M, Watanabe G, Daidone MG, Roayaie S, Schwartz M, Thung S, Salvesen HB, Gabriel S, Mazzaferro V, Bruix J, Friedman SL, Kumada H, Llovet JM, Golub TR. Gene expression in fixed tissues and outcome in hepatocellular carcinoma. N Engl J Med 2008; 359(19): 1995-2004

[28] Jiang J, Gusev Y, Aderca I, Mettler TA, Nagorney DM, Brackett DJ, Roberts LR, Schmittgen TD. Association of MicroRNA expression in hepatocellular carcinomas with hepatitis infection, cirrhosis, and patient survival. Clin Cancer Res 2008; 14(2): 419-427

[29] Lou C, Du Z, Yang B, Gao Y, Wang Y, Fang S. Aberrant DNA methylation profile of hepatocellular carcinoma and surgically resected margin. Cancer Sci 2009; 100(6): 996-1004

[30] Villanueva A, Hoshida Y, Toffanin S, Lachenmayer A, Alsinet C, Savic R, Cornella H, Llovet JM. New strategies in hepatocellular carcinoma: genomic prognostic markers. Clin Cancer Res 2010; 16(19): 4688-4694

[31] Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, Schwartz M, Porta C, Zeuzem S, Bolondi L, Greten TF, Galle PR, Seitz JF, Borbath I, Häussinger D, Giannaris T, Shan M, Moscovici M, Voliotis D, Bruix J, the SISG. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008; 359(4): 378-390

[32] Villanueva A, Llovet JM. Targeted therapies for hepatocellular carcinoma. Gastroenterology 2011; 140(5): 1410-1426

[33] Ribatti D, Vacca A, Nico B, Sansonno D, Dammacco F. Angiogenesis and anti-angiogenesis in hepatocellular carcinoma. Cancer Treat Rev 2006; 32(6): 437-444

[34] Zhu AX, Blaszkowsky LS, Ryan DP, Clark JW, Muzikansky A, Horgan K, Sheehan S, Hale KE, Enzinger PC, Bhargava P, Stuart K. Phase II study of gemcitabine and oxaliplatin in combination with bevacizumab in patients with advanced hepatocellular carcinoma. J Clin Oncol 2006; 24(12): 1898-1903

[35] Yoshiji H, Kuriyama S, Yoshii J, Ikenaka Y, Noguchi R, Hicklin DJ, Huber J, Nakatani T, Tsujinoue H, Yanase K, Imazu H, Fukui H. Synergistic effect of basic fibroblast growth factor and vascular endothelial growth factor in murine hepatocellular carcinoma. Hepatology 2002; 35(4): 834-842

[36] Thomas MB, Chadha R, Glover K, Wang X, Morris J, Brown T, Rashid A, Dancey J, Abbruzzese JL. Phase 2 study of erlotinib in patients with unresectable hepatocellular carcinoma. Cancer 2007; 110(5): 1059-1067

[37] Ramanathan RK, Belani CP, Singh DA, Tanaka M, Lenz HJ, Yen Y, Kindler HL, Iqbal S, Longmate J, Mack PC, Lurje G, Gandour-Edwards R, Dancey J, Gandara DR. A phase II study of lapatinib in patients with advanced biliary tree and hepatocellular cancer. Cancer Chemother Pharmacol 2009; 64(4): 777-783

[38] Höpfner M, Sutter AP, Huether A, Schuppan D, Zeitz M, Scherübl H. Targeting the epidermal growth factor receptor by gefitinib for treatment of hepatocellular carcinoma. J Hepatol 2004; 41(6): 1008-1016

[39] Fuchs BC, Fujii T, Dorfman JD, Goodwin JM, Zhu AX, Lanuti M, Tanabe KK. Epithelial-to-mesenchymal transition and integrin-linked kinase mediate sensitivity to epidermal growth factor receptor inhibition in human hepatoma cells. Cancer Res 2008; 68(7): 2391-2399

[40] Comoglio PM, Giordano S, Trusolino L. Drug development of MET inhibitors: targeting oncogene addiction and expedience. Nat Rev Drug Discov 2008; 7(6): 504-516

[41] Villanueva A, Chiang DY, Newell P, Peix J, Thung S, Alsinet C, Tovar V, Roayaie S, Minguez B, Sole M, Battiston C, Van Laarhoven S, Fiel MI, Di Feo A, Hoshida Y, Yea S, Toffanin S, Ramos A, Martignetti JA, Mazzaferro V, Bruix J, Waxman S, Schwartz M, Meyerson M, Friedman SL, Llovet JM. Pivotal role of mTOR signaling in hepatocellular carcinoma. Gastroenterology 2008;135(6): 1972-1983. e1-11

[42] Huynh H, Chow KH, Soo KC, Toh HC, Choo SP, Foo KF, Poon D, Ngo VC, Tran E. RAD001 (everolimus) inhibits tumour growth in xenograft models of human hepatocellular carcinoma. J Cell Mol Med 2009; 13(7): 1371-1380

[43] Zhu AX, Abrams TA, Miksad R, Blaszkowsky LS, Meyerhardt JA, Zheng H, Muzikansky A, Clark JW, Kwak EL, Schrag D, Jors KR, Fuchs CS, Iafrate AJ, Borger DR, Ryan DP. Phase 1/2 study of everolimus in advanced hepatocellular carcinoma. Cancer 2011;117(22):5094-5102

[44] Newell P, Toffanin S, Villanueva A, Chiang DY, Minguez B, Cabellos L, Savic R, Hoshida Y, Lim KH, Melgar-Lesmes P, Yea S, Peix J, Deniz K, Fiel MI, Thung S, Alsinet C, Tovar V, Mazzaferro V, Bruix J, Roayaie S, Schwartz M, Friedman SL, Llovet JM. Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo. J Hepatol 2009; 51(4): 725-733

[45] Wang Z, Zhou J, Fan J, Tan CJ, Qiu SJ, Yu Y, Huang XW, Tang ZY. Sirolimus inhibits the growth and metastatic progression of hepatocellular carcinoma. J Cancer Res Clin Oncol 2009; 135(5): 715-722

[46] Llovet JM, Bruix J. Molecular targeted therapies in hepatocellular carcinoma. Hepatology 2008; 48(4): 1312-1327

[47] Shi YH, Ding WX, Zhou J, He JY, Xu Y, Gambotto AA, Rabinowich H, Fan J, Yin XM. Expression of X-linked inhibitor-of-apoptosis protein in hepatocellular carcinoma promotes metastasis and tumor recurrence. Hepatology 2008; 48(2): 497-507

[48] Ding ZB, Shi YH, Zhou J, Qiu SJ, Xu Y, Dai Z, Shi GM, Wang XY, Ke AW, Wu B, Fan J. Association of autophagy defect with a malignant phenotype and poor prognosis of hepatocellular carcinoma. Cancer Res 2008; 68(22): 9167-9175

[49] Shi YH, Ding ZB, Zhou J, Qiu SJ, Fan J. Prognostic significance of Beclin 1-dependent apoptotic activity in hepatocellular carcinoma. Autophagy 2009; 5(3): 380-382

[50] Yang JD, Nakamura I, Roberts LR. The tumor microenvironment in hepatocellular carcinoma: current status and therapeutic targets. Semin Cancer Biol 2011; 21(1): 35-43

[51] Mazzocca A, Fransvea E, Lavezzari G, Antonaci S, Giannelli G. Inhibition of transforming growth factor beta receptor I kinase blocks hepatocellular carcinoma growth through neo-angiogenesis regulation. Hepatology 2009; 50(4): 1140-1151

[52] Fransvea E, Mazzocca A, Antonaci S, Giannelli G. Targeting transforming growth factor (TGF)-betaRI inhibits activation of beta1 integrin and blocks vascular invasion in hepatocellular carcinoma. Hepatology 2009; 49(3): 839-850

[53] Fransvea E, Angelotti U, Antonaci S, Giannelli G. Blocking transforming growth factor-beta up-regulates E-cadherin and reduces migration and invasion of hepatocellular carcinoma cells. Hepatology 2008; 47(5): 1557-1566

[54] Ke AW, Shi GM, Zhou J, Huang XY, Shi YH, Ding ZB, Wang XY, Devbhandari RP, Fan JCD151 amplifies signaling by integrin alpha6beta1 to PI3K and induces the epithelial-mesenchymal transition in HCC cells. Gastroenterology 2011;140(5):1629-1641. e15

[55] Shi GM, Ke AW, Zhou J, Wang XY, Xu Y, Ding ZB, Devbhandari RP, Huang XY, Qiu SJ, Shi YH, Dai Z, Yang XR, Yang GH, Fan J. CD151 modulates expression of matrix metalloproteinase 9 and promotes neoangiogenesis and progression of hepatocellular carcinoma. Hepatology 2010; 52(1): 183-196

[56] Ke AW, Shi GM, Zhou J, Wu FZ, Ding ZB, Hu MY, Xu Y, Song ZJ, Wang ZJ, Wu JC, Bai DS, Li JC, Liu KD, Fan J. Role of overexpression of CD151 and/or c-Met in predicting prognosis of hepatocellular carcinoma. Hepatology 2009; 49(2): 491-503

[57] Gao Y, Yao A, Zhang W, Lu S, Yu Y, Deng L, Yin A, Xia Y, Sun B, Wang X. Human mesenchymal stem cells overexpressing pigment epithelium-derived factor inhibit hepatocellular carcinoma in nude mice. Oncogene 2010; 29(19): 2784-2794

[58] Li YW, Qiu SJ, Fan J, Zhou J, Gao Q, Xiao YS, Xu YF. Intratumoral neutrophils: a poor prognostic factor for hepatocellular carcinoma following resection. J Hepatol 2011; 54(3): 497-505

[59] Kuang DM, Zhao Q, Wu Y, Peng C, Wang J, Xu Z, Yin XY, Zheng L. Peritumoral neutrophils link inflammatory response to disease progression by fostering angiogenesis in hepatocellular carcinoma. J Hepatol 2011; 54(5): 948-955

[60] Zhu XD, Zhang JB, Zhuang PY, Zhu HG, Zhang W, Xiong YQ, Wu WZ, Wang L, Tang ZY, Sun HC. High expression of macrophage colony-stimulating factor in peritumoral liver tissue is associated with poor survival after curative resection of hepatocellular carcinoma. J Clin Oncol 2008; 26(16): 2707-2716

[61] Gao Q, Qiu SJ, Fan J, Zhou J, Wang XY, Xiao YS, Xu Y, Li YW, Tang ZY. Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection. J Clin Oncol 2007; 25(18): 2586-2593

[62] Gao Q, Zhou J, Wang XY, Qiu SJ, Song K, Huang XW, Sun J, Shi YH, Li BZ, Xiao YS, Fan J. Infiltrating memory/Senescent T cell ratio predicts extrahepatic metastasis of hepatocellular carcinoma. Ann Surg Oncol 2012;19(2):455-466

[63] Takayama T, Sekine T, Makuuchi M, Yamasaki S, Kosuge T, Yamamoto J, Shimada K, Sakamoto M, Hirohashi S, Ohashi Y, Kakizoe T. Adoptive immunotherapy to lower postsurgical recurrence rates of hepatocellular carcinoma: a randomised trial. Lancet 2000; 356(9232): 802-807

[64] Greten TF, Manns MP, Korangy F. Immunotherapy of hepatocellular carcinoma. J Hepatol 2006; 45(6): 868-878

[65] Llovet JM, Sala M, Castells L, Suarez Y, Vilana R, Bianchi L, Ayuso C, Vargas V, Rodés J, Bruix J. Randomized controlled trial of interferon treatment for advanced hepatocellular carcinoma. Hepatology 2000; 31(1): 54-58

[66] Ikeda K, Arase Y, Saitoh S, Kobayashi M, Suzuki Y, Suzuki F, Tsubota A, Chayama K, Murashima N, Kumada H. Interferon beta prevents recurrence of hepatocellular carcinoma after complete resection or ablation of the primary tumor-A prospective randomized study of hepatitis C virus-related liver cancer. Hepatology 2000; 32(2): 228-232

[67] Kubo S, Nishiguchi S, Hirohashi K, Tanaka H, Shuto T, Yamazaki O, Shiomi S, Tamori A, Oka H, Igawa S, Kuroki T, Kinoshita H. Effects of long-term postoperative interferon-alpha therapy on intrahepatic recurrence after resection of hepatitis C virus-related hepatocellular carcinoma. A randomized, controlled trial. Ann Intern Med 2001; 134(10): 963-967

[68] Reinisch W, Holub M, Katz A, Herneth A, Lichtenberger C, Schoniger-Hekele M, Waldhoer T, Oberhuber G, Ferenci P, Gangl A, Mueller C. Prospective pilot study of recombinant granulocyte-macrophage colony-stimulating factor and interferon-gamma in patients with inoperable hepatocellular carcinoma. J Immunother 2002; 25(6): 489-499

[69] Palmieri G, Montella L, Milo M, Fiore R, Biondi E, Bianco AR, Martignetti A. Ultra-low-dose interleukin-2 in unresectable hepatocellular carcinoma. Am J Clin Oncol 2002; 25(3): 224-226

[70] Iwashita Y, Tahara K, Goto S, Sasaki A, Kai S, Seike M, Chen CL, Kawano K, Kitano S. A phase I study of autologous dendritic cell-based immunotherapy for patients with unresectable primary liver cancer. Cancer Immunol Immunother 2003; 52(3): 155-161

[71] Patt YZ, Hassan MM, Lozano RD, Brown TD, Vauthey JN, Curley SA, Ellis LM. Phase II trial of systemic continuous fluorouracil and subcutaneous recombinant interferon Alfa-2b for treatment of hepatocellular carcinoma. J Clin Oncol 2003; 21(3): 421-427

[72] Feun LG, O’Brien C, Molina E, Rodriguez M, Jeffers L, Schiff ER, Marini A, Savaraj N, Ardalan B. Recombinant leukocyte interferon, doxorubicin, and 5FUDR in patients with hepatocellular carcinoma-A phase II trial. J Cancer Res Clin Oncol 2003; 129(1): 17-20

[73] Kuang M, Peng BG, Lu MD, Liang LJ, Huang JF, He Q, Hua YP, Totsuka S, Liu SQ, Leong KW, Ohno T. Phase II randomized trial of autologous formalin-fixed tumor vaccine for postsurgical recurrence of hepatocellular carcinoma. Clin Cancer Res 2004; 10(5): 1574-1579

[74] Shi M, Zhang B, Tang ZR, Lei ZY, Wang HF, Feng YY, Fan ZP, Xu DP, Wang FS. Autologous cytokine-induced killer cell therapy in clinical trial phase I is safe in patients with primary hepatocellular carcinoma. World J Gastroenterol 2004; 10(8): 1146-1151

[75] Chen S, Li B, Xie H, Xu L, Niu G, Fan K, Fan Q. Phase I clinical trial of targeted therapy using ¹³¹I-Hepama-1 mAb in patients with hepatocellular carcinoma. Cancer Biother Radiopharm 2004; 19(5): 589-600

[76] Lee WC, Wang HC, Hung CF, Huang PF, Lia CR, Chen MF. Vaccination of advanced hepatocellular carcinoma patients with tumor lysate-pulsed dendritic cells: a clinical trial. J Immunother 2005; 28(5): 496-504

[77] Yin XY, Lü MD, Liang LJ, Lai JM, Li DM, Kuang M. Systemic chemo-immunotherapy for advanced-stage hepatocellular carcinoma. World J Gastroenterol 2005; 11(16): 2526-2529

[78] Chi KH, Liu SJ, Li CP, Kuo HP, Wang YS, Chao Y, Hsieh SL. Combination of conformal radiotherapy and intratumoral injection of adoptive dendritic cell immunotherapy in refractory hepatoma. J Immunother 2005; 28(2): 129-135

[79] Butterfield LH, Ribas A, Dissette VB, Lee Y, Yang JQ, De la Rocha P, Duran SD, Hernandez J, Seja E, Potter DM, McBride WH, Finn R, Glaspy JA, Economou JS. A phase I/II trial testing immunization of hepatocellular carcinoma patients with dendritic cells pulsed with four alpha-fetoprotein peptides. Clin Cancer Res 2006; 12(9): 2817-2825

[80] Chen ZN, Mi L, Xu J, Song F, Zhang Q, Zhang Z, Xing JL, Bian HJ, Jiang JL, Wang XH, Shang P, Qian AR, Zhang SH, Li L, Li Y, Feng Q, Yu XL, Feng Y, Yang XM, Tian R, Wu ZB, Leng N, Mo TS, Kuang AR, Tan TZ, Li YC, Liang DR, Lu WS, Miao J, Xu GH, Zhang ZH, Nan KJ, Han J, Liu QG, Zhang HX, Zhu P. Targeting radioimmunotherapy of hepatocellular carcinoma with iodine (¹³¹I) metuximab injection: clinical phase I/II trials. Int J Radiat Oncol Biol Phys 2006; 65(2): 435-444

[81] Mazzaferro V, Romito R, Schiavo M, Mariani L, Camerini T, Bhoori S, Capussotti L, Calise F, Pellicci R, Belli G, Tagger A, Colombo M, Bonino F, Majno P, Llovet JM. Prevention of hepatocellular carcinoma recurrence with alpha-interferon after liver resection in HCV cirrhosis. Hepatology 2006; 44(6): 1543-1554

[82] Sun HC, Tang ZY, Wang L, Qin LX, Ma ZC, Ye QH, Zhang BH, Qian YB, Wu ZQ, Fan J, Zhou XD, Zhou J, Qiu SJ, Shen YF. Postoperative interferon alpha treatment postponed recurrence and improved overall survival in patients after curative resection of HBV-related hepatocellular carcinoma: a randomized clinical trial. J Cancer Res Clin Oncol 2006; 132(7): 458-465

[83] Lo CM, Liu CL, Chan SC, Lam CM, Poon RT, Ng IO, Fan ST, Wong J. A randomized, controlled trial of postoperative adjuvant interferon therapy after resection of hepatocellular carcinoma. Ann Surg 2007; 245(6): 831-842

[84] Kudo M, Sakaguchi Y, Chung H, Hatanaka K, Hagiwara S, Ishikawa E, Takahashi S, Kitai S, Inoue T, Minami Y, Ueshima K. Long-term interferon maintenance therapy improves survival in patients with HCV-related hepatocellular carcinoma after curative radiofrequency ablation. A matched case-control study. Oncology 2007; 72(Suppl 1): 132-138

[85] Weng DS, Zhou J, Zhou QM, Zhao M, Wang QJ, Huang LX, Li YQ, Chen SP, Wu PH, Xia JC. Minimally invasive treatment combined with cytokine-induced killer cells therapy lower the short-term recurrence rates of hepatocellular carcinomas. J Immunother 2008; 31(1): 63-71

[86] Dollinger MM, Behrens CM, Lesske J, Behl S, Behrmann C, Fleig WE. Thymostimulin in advanced hepatocellular carcinoma: a phase II trial. BMC Cancer 2008; 8(1): 72

[87] Dong H, Li Q, Wang J, Zhang T, Kong DL. A randomized, controlled trial of postoperative adjuvant cytokine-induced killer cells immunotherapy after radical resection of hepatocellular carcinoma. Dig Liver Dis 2009; 41(1): 36-41

[88] Palmer DH, Midgley RS, Mirza N, Torr EE, Ahmed F, Steele JC, Steven NM, Kerr DJ, Young LS, Adams DH. A phase II study of adoptive immunotherapy using dendritic cells pulsed with tumor lysate in patients with hepatocellular carcinoma. Hepatology 2009; 49(1): 124-132

[89] Greten TF, Ormandy LA, Fikuart A, Höchst B, Henschen S, Hörning M, Manns MP, Korangy F. Low-dose cyclophosphamide treatment impairs regulatory T cells and unmasks AFP-specific CD4⁺ T-cell responses in patients with advanced HCC. J Immunother 2010; 33(2): 211-218

[90] Yoshikawa T, Nakatsugawa M, Suzuki S, Shirakawa H, Nobuoka D, Sakemura N, Motomura Y, Tanaka Y, Hayashi S, Nakatsura T. HLA-A2-restricted glypican-3 peptide-specific CTL clones induced by peptide vaccine show high avidity and antigen-specific killing activity against tumor cells. Cancer Sci 2011; 102(5): 918-925

[91] Breous E, Thimme R. Potential of immunotherapy for hepatocellular carcinoma. J Hepatol 2011; 54(4): 830-834

[92] Gao Q, Wang XY, Qiu SJ, Yamato I, Sho M, Nakajima Y, Zhou J, Li BZ, Shi YH, Xiao YS, Xu Y, Fan J. Overexpression of PD-L1 significantly associates with tumor aggressiveness and postoperative recurrence in human hepatocellular carcinoma. Clin Cancer Res 2009; 15(3): 971-979

[93] Cai MY, Xu YF, Qiu SJ, Ju MJ, Gao Q, Li YW, Zhang BH, Zhou J, Fan J. Human leukocyte antigen-G protein expression is an unfavorable prognostic predictor of hepatocellular carcinoma following curative resection. Clin Cancer Res 2009; 15(14): 4686-4693

[94] Eyler CE, Rich JN. Survival of the fittest: cancer stem cells in therapeutic resistance and angiogenesis. J Clin Oncol 2008; 26(17): 2839-2845

[95] Shi GM, Xu Y, Fan J, Zhou J, Yang XR, Qiu SJ, Liao Y, Wu WZ, Ji Y, Ke AW, Ding ZB, He YZ, Wu B, Yang GH, Qin WZ, Zhang W, Zhu J, Min ZH, Wu ZQ. Identification of side population cells in human hepatocellular carcinoma cell lines with stepwise metastatic potentials. J Cancer Res Clin Oncol 2008; 134(11): 1155-1163

[96] Haraguchi N, Ishii H, Mimori K, Tanaka F, Ohkuma M, Kim HM, Akita H, Takiuchi D, Hatano H, Nagano H, Barnard GF, Doki Y, Mori M. CD13 is a therapeutic target in human liver cancer stem cells. J Clin Invest 2010; 120(9): 3326-3339

[97] Yamashita T, Ji J, Budhu A, Forgues M, Yang W, Wang HY, Jia H, Ye Q, Qin LX, Wauthier E, Reid LM, Minato H, Honda M, Kaneko S, Tang ZY, Wang XW. EpCAM-positive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features. Gastroenterology 2009; 136(3): 1012-1024

[98] Yang ZF, Ho DW, Ng MN, Lau CK, Yu WC, Ngai P, Chu PW, Lam CT, Poon RT, Fan ST. Significance of CD90⁺ cancer stem cells in human liver cancer. Cancer Cell 2008; 13(2): 153-166

[99] Yin S, Li J, Hu C, Chen X, Yao M, Yan M, Jiang G, Ge C, Xie H, Wan D, Yang S, Zheng S, Gu J. CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity. Int J Cancer 2007; 120(7): 1444-1450

[100] Yamashita T, Honda M, Nio K, Nakamoto Y, Yamashita T, Takamura H, Tani T, Zen Y, Kaneko S. Oncostatin m renders epithelial cell adhesion molecule-positive liver cancer stem cells sensitive to 5-Fluorouracil by inducing hepatocytic differentiation. Cancer Res 2010; 70(11): 4687-4697

[101] Mishra L, Banker T, Murray J, Byers S, Thenappan A, He AR, Shetty K, Johnson L, Reddy EP. Liver stem cells and hepatocellular carcinoma. Hepatology 2009; 49(1): 318-329

[102] Yang XR, Xu Y, Yu B, Zhou J, Qiu SJ, Shi GM, Zhang BH, Wu WZ, Shi YH, Wu B, Yang GH, Ji Y, Fan J. High expression levels of putative hepatic stem/progenitor cell biomarkers related to tumour angiogenesis and poor prognosis of hepatocellular carcinoma. Gut 2010; 59(7): 953-962

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