Novel biomarkers in hepatocellular carcinoma

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Contents lists available at ScienceDirect

Digestive and Liver Disease journal homepage: www.elsevier.com/locate/dld

Review Article

Novel biomarkers in hepatocellular carcinoma Felice De Stefano, Eduardo Chacon, Lilia Turcios, Francesc Marti, Roberto Gedaly ∗ Transplant and Hepatobiliary Center, Department of Surgery, University of Kentucky College of Medicine, Lexington, KY, United States

a r t i c l e

i n f o

Article history: Received 13 June 2018 Received in revised form 9 August 2018 Accepted 13 August 2018 Available online xxx Keywords: Biomarkers Circular RNAs Hepatocellular carcinoma Micro RNAs Tumor microenvironment

a b s t r a c t Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths and the fifth most common cancer worldwide. Most of these patients are seen with advanced disease at the time of presentation. In spite of its high prevalence, there are not many therapeutic options available for patients with advanced-stage HCC. There is an urgent need for improving early detection and prognostication of patients with HCC. In addition, the development of new therapies targeting specific pathways involved in the pathogenesis of HCC should be a major goal for future research, with the objective of improving outcomes of patients with HCC. Biomarkers represent a relatively easy and noninvasive way to detect and estimate disease prognosis. In spite of the numerous efforts to find molecules as possible biomarkers, there is not a single ideal marker in HCC. Many new findings have shown promising results both in diagnosing and treating HCC. In this review, we summarized the most recent and relevant biomarkers in HCC. © 2018 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l.

1. Introduction Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths and the fifth most common cancer worldwide [1,2]. Chronic inflammatory diseases affecting the liver such as cirrhosis, hepatitis B and C virus infection, alcoholic liver disease and non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH) are considered major risk factors for HCC [3–6]. Carcinogens such as nitrites, hydrocarbons, aflatoxins, organochlorine pesticides and genetic disorders such as hemochromatosis, Wilsons disease and alpha-1 antitrypsin deficiency are also associated with the development of HCC [7,8]. Over the past decades, the incidence and deaths associated with HCC have increased at great scale. The overall 5-year survival of patients with liver cancer is currently 10–20% [9]. However, early diagnosis may remarkably improve the prognosis. HCC surveillance guidelines recommend the use of imaging methods such as CT scan, MRI or ultrasound (US) alone or in combination with the measurement of serum alpha-fetoprotein (AFP) levels to diagnose HCC in cirrhotics [10]. AFP is the most widely accepted diagnostic serum biomarker. However, its low sensitivity and specificity limit its clinical use [11]. A significant proportion of HCC patients do not have elevation of serum AFP levels as described

∗ Corresponding author at: University of Kentucky Transplant Center, 800 Rose Street, C451, Lexington, KY 40536-0293, United States. E-mail address: [email protected] (R. Gedaly).

by Agopian et al. in a recent study in which they reported 31.3% of non-AFP-producing tumors in a cohort of 665 patients [12]. There have been multiple attempts to use combinations of different biomarkers to improve the sensitivity, specificity and predictive value of single markers. Recently, the Japan Society of Hepatology (JSH) guidelines added to their recommendations the use of AFP in combination with des-␥-carboxyprothrombin (DCP) and the Lens culinaris agglutinin (AFP-L3) [13]. Other groups have proposed the use of biomarker combinations together with demographic information to enhance the diagnostic accuracy. The GALAD score uses patient’s age and gender in conjunction with AFP, DCP and AFP-L3 levels [14]. The American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) guidelines includes CD34, CK7, glypican 3, HSP-70, and glutamine synthetase staining to improve diagnostic accuracy [15] while the EASL adds gene expression profiles of GPC3 and survivin [16]. Regarding prognosis EASL recommends the use of AFP levels, VEGF and Angiopoietin 2 as independent prognostic biomarkers, in addition to the possible implementation of keratin 19 and EpCAM because of their correlation with worse outcomes in patients with HCC [16]. The aim of this review is to perform a comprehensive overview of the most relevant HCC biomarkers currently in the published literature, specially focused on the association with cancer development and their potential use as diagnostic, prognostic and therapeutic targets in HCC.

https://doi.org/10.1016/j.dld.2018.08.019 1590-8658/© 2018 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l.

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2. Molecular and biochemical cellular markers 2.1. Glypican-3 Glypican-3 (GPC3) is a member of the glypican family of heparan sulfate proteoglycans. Its main role is to regulate developmental morphogenesis by growth factors through the Wnt and Hedgehog cell signaling pathways [3,17]. A low expression of GPC3 mRNA has been found in normal adult heart, lung, kidney and ovarian tissues. GPC3 can be also be detected in serum, where it is released by the lipase Notum [18]. Several studies using different detection techniques have found low or absent expression of GPC3 in normal liver tissue, focal nodular hyperplasia (FNH) and hepatocellular adenoma (HA), whereas the majority of HCC tissues overexpressed GPC3 [19,20]. One study demonstrated a higher GPC3 expression in small HCCs than in cirrhotic samples and other small focal lesions, thus suggesting the acute raise of GPC3 expression in the transition of premalignant nodules to HCC [21]. GPC3 is also occasionally expressed in other types of tumors, such as liposarcoma, testicular non-seminomatous germ cell tumors and squamous cell carcinoma of the lung [22]. Studies have revealed the potential diagnostic value of GPC3 as a serum marker in HCC [23]. In fact, serum GPC3 showed higher specificity and sensitivity than Human-Cervical-Cancer-Oncogene (HCCR) and AFP to diagnose HCC, and the combination of the three markers demonstrated higher sensitivity than any of the markers alone [24]. In addition, GPC3 tumor levels have been correlated with higher recurrence rates and decreased overall and disease-free survival in several clinical studies [25,26]. The role of GPC3 as a targeted therapy is promising. Two clinical trials in USA and Japan are testing GC33, the first humanized mouse anti-GPC3 antibody for advanced HCC. In both trials, GC33 has been well tolerated and it has showed preliminary antitumor activity, with accumulation of infiltrating cytotoxic T-lymphocytes (CTLs) into tumor tissues and an increased antibody-dependent cell cytotoxic activity [3,27,28]. Two additional anti-GPC3 antibodies are currently being tested in preclinical studies: HN3, a human single domain antibody that can directly inhibit HCC cell proliferation [29], and MDX-1414 [3]. Another attractive option for HCC treatment is the use of antigen-specific cancer immunotherapies such as peptide vaccines, dendritic cell vaccines and adoptive cell transference. Phase I and II clinical trials demonstrated a significant correlation between the induction of peptide-specific CTLs and patient survival [30,31]. In a phase II study with HCC patients who underwent surgery or radiofrequency ablation (RFA) therapy, the patients that received GPC3-derived peptide vaccine as adjuvant therapy showed a significant decrease in 1-year recurrence rate compared to those treated with surgery alone [31] (Table 1). 2.2. Osteopontin Osteopontin (OPN) is an extracellular matrix multifunctional protein that participates in several biological processes [32,33]. In physiological conditions OPN is expressed in some liver cells such as Kupffer and stellate cells, but not in hepatocytes [2]. Remarkably, the depletion of OPN in HCC cell lines resulted in the decreased expression of stem-cell associated genes, side population cells fraction, formation of hepato-spheroids and tumorigenecity in immune deficient mice [34]. In concordance, depletion of OPN attenuated hepatocarcinogenesis in mice [35]. Studies have reported the upregulation of OPN in several types of malignancies, including lung, breast, colon and HCC. An interesting study by Duarte-Salles et al. demonstrated the association between pre-diagnostic circulating OPN levels and HCC incidence in a large cohort. Addition of AFP to the OPN levels improved the

prediction potential in this cohort [36]. In concordance with these results, other studies showed that the combination of OPN and AFP increased the sensitivity and specificity for the diagnosis of HCC compared to each of these markers alone [37,38]. It has been proposed that OPN is required to maintain the stemlike properties in HCC cells. Cao et al. demonstrated a significant correlation between the expression levels of OPN in the cell localized at the edge of the tumor and the clinical prognosis of patients with HCC. Overall, this evidence supports the importance of OPN in HCC development and progression and highlights its potential as diagnostic and prognostic marker. 2.3. Des--carboxyprothrombin Des-gamma carboxyprothrombin (DCP) is also known as prothrombin induced by vitamin K absence-II (PIVKA II). DCP is a nonfunctional precursor of prothrombin originated from an impaired vitamin K mediated post-translational ␥-carboxylation of glutamic acid residues. An excessive synthesis of prothrombin precursors by HCC cells will raise DCP levels, which several studies have associated with the progression of the disease and tumor diameter [39–43] along with the development of portal vein invasion (PVI), a strong negative prognostic indicator in HCC [39]. Likely because of the structural resemblance with hepatocyte growth factor, DCP has been recently identified as an autologous growth factor in the stimulation of HCC proliferation [31], and as a paracrine factor in the integration of HCC with vascular endothelial cells [44]. DCP may promote angiogenesis via activation of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) [45,46]. A study comparing the serum levels of four HCC biomarkers (DCP, GP73, AFP, AFP-L3) revealed that for early stage diagnosis, DCP has 60% sensitivity and 64.5% specificity and for all stages HCC 62.5% and 85.5%, respectively. They found that combination of the four serum biomarkers is more accurate than any of them alone to diagnose HCC [47]. Overall sensitivity of 67% and 71% and specificity of 92% and 84% for HCC diagnosis have been reported [48–50]. Lai et al. demonstrated in a recent meta-analysis a 5-fold increased risk of recurrence after liver transplantation for HCC in patients with high levels of DCP [51]. 2.4. AFP-L3 ␣-Fetoprotein (AFP) is a protein produced in the liver during fetal development and it is thought to be the fetal analog of serum albumin. Alpha-fetoprotein (AFP) is the principal serum AFP-glycoform found in HCC patients. The glycosylated form of serum AFP that is most closely associated with HCC is designated as AFP-L3. The simultaneous determination of AFP-L3, AFP with p53 antigen [52] or with DCP [53] showed increased diagnostic accuracy and sensitivity than any of the three markers alone. In the DCP study, the authors proposed the “GALAD score”, which incorporates patient’s gender and age to the serological biomarkers of AFP-L3, AFP and DCP to increase the sensitivity and specificity of the algorithm and its clinical effectiveness in the early recognition of HCC. In fact, the GALAD algorithm provided the highest AUC (0.9242) in the BCLC 0/A stage HCC cohort [54]. 2.5. Golgi protein-73 Golgi protein-73 (GP73) is a 73-kDa type-II Golgi transmembrane glycoprotein. It promotes HCC cell invasion through activation of a cAMP responsive element binding protein (CREB)-mediated transcription, which enhances the matrix metalloproteinase-13 (MMP-13) expression [55]. GP73 overexpression increased proliferation, migration of HCC cell lines and xenograft tumor growth in mice in an mTORC1-dependent pro-

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Table 1 HCC biomarkers and their clinical application. Name of biomarker

Clinical potential application

Validation method: prospective/retrospective/cross-sectional, single-center vs multi-center

Reference

Molecular and biochemical cellular markers Glypican-3 (GPC3) Osteopontin (OPN) Des-␥-carboxyprothrombin (DCP) AFP-L3 Golgi protein-73 (GP73)

Diagnostic and prognostic Diagnostic and prognostic Diagnostic and prognostic Diagnostic and prognostic Diagnostic and prognostic

Prospective, multi-centera Prospective, multi-centera Prospective, single-centera Retrospective, single-center Prospective, single-center

[3,17–31] [2,32–38] [31,39–51] [38,53,54] [55–61]

Cancer stem cell markers CD44 CD133 CD90 EpCAM

Prognostic Prognostic. Associated to drug resistance Prognostic. Associated to drug resistance Therapeutic potential target

Pre-clinical evidence Pre-clinical evidence Retrospective, single-center Retrospective, multi-center

[78,97] [98] [99–101] [102–104]

Tumor stroma/tumor microenvironment Cellular components Cancer associated fibroblasts (CAFs) Hepatic stellate cells (HSCs) Tumor associated macrophages (TAMs) Lymphocytes

Prognostic. Associated to tumor invasion Prognostic. Associated to tumor invasion Prognostic Prognostic

Pre-clinical evidence Pre-clinical evidence Retrospective, single-center Meta-analysis

[17,105,106] [105,107,108] [17,105,109] [107,110]

Prognostic. Therapeutic potential target Prognostic. Associated to drug resistance Prognostic Prognostic. Therapeutic target

Cross-sectional, multi-center Pre-clinical evidence Review Cross-sectional, single-center

[107,111,112] [107,113] [114] [115]

Non-cellular components Transforming growth factor-beta (TGF-␤) Fibroblast growth factor (FGF) Hepatocyte growth factor/scatter factor (HGF/SF) Vascular endothelial growth factor (VEGF) a

Biomarker with additional retrospective/single-center evidence.

cess. In a recent article, Yang et al. reported that GP73 promoted epithelial mesenchymal transition (EMT) and HCC cell invasion associated with increased TGF-␤1-dependent levels of p-Smad2pSmad2. Analysis of the clinical HCC samples revealed a positive correlation between GP73 and TGF-␤1-dependent Smad2 [56]. Interestingly, high expression of GP73 is present in focal nodular hyperplasia (FNH) [57], demonstrating that it can be expressed in both benign and malignant liver diseases. Several meta-analysis support the high sensitivity and specificity of serum GP73 expression as potential diagnostic biomarker for HCC, showing even better scores than those seen with AFP levels [58]. Still, a significant improvement was observed after combining both markers [59]. For AFP-negative HCC patients there is a lack of efficient and reliable diagnostic methods. Interestingly, 60% of AFP-negative HCC patients tested positive for GP73, although the sensitivity and diagnostic accuracy in those patients was lower than those scored in GP73-positive AFP-L3-positive patients [60,61]. Furthermore, incubation with the mTORC1-inhibitor rapamycin decreased the GP73 expression in HCC and other cell lines [56], suggesting the potential therapeutic benefit of mTOR inhibitors to arrest HCC proliferation and invasion through the inhibition of GP73 expression. Although the diagnostic potential of serum GP73 requires further investigation, it appears to be a suitable biomarker for prognosis, and the GP73-CREB-MMP-13 axis may become a promising target for HCC therapy.

2.6. MicroRNAs MicroRNAs (miRNAs) are short, single-stranded, non-coding endogenous RNA molecules that regulate gene expression by targeting messenger RNA (mRNA) for cleavage or translational repression [62,63]. miRNAs play a critical role in cell development, proliferation and apoptosis, which are crucial processes for carcinogenesis and tumor progression. miRNAs may participate in the diverse cancer processes through the regulation of cell death resistance, insensitivity to antigrowth signals, avoidance of immune destruction, tissue

invasion/metastasis, tumor promoted inflammation, angiogenesis, genome instability and mutations [62]. Several studies have described the association between abnormal levels of miRNAs expression and, clinico-pathological characteristics, and prognosis in several malignancies such as breast cancer, prostate cancer, lung cancer and HCC [64–66]. Thus, there is a significant interest to study the impact of miRNAs in cancer development and the potential use as biomarkers. Up- or down-regulation of different miRNAs can play an important role in HCC. Some miRNAs (such as miR-122) are normally expressed in the healthy liver tissue but the liver-specific miR122 is frequently suppressed in primary HCC tumors [67]. The downregulation of miR-122 in HCC-derived cell lines enhanced proliferation, colony formation, cell invasion and promoted EMT, whereas upregulation of miR-122 caused the opposite effects [68,69], and several studies proposed that miR-122 may act as a tumor suppressor [68,70,71]. Importantly, this micro-RNA can regulate the Wnt signaling pathway [72], which dysregulation in many cancers (including HCC) is associated with tumor proliferation, invasion and progression. In fact, low expression of miR-122 in HCC patients correlates with poor prognosis [68]. A study by Mohamed et al., found that circulating miR-23a is associated with multiple hepatic focal lesions. They also found that a cutoff value of ≥210 miR-23a was significantly more sensitive, specific and accurate to diagnose HCC than using AFP cut off level of ≥200 ng/ml [73]. They concluded that miR-23a has a potential role in screening or as a prognostic biomarker for HCC patients. Other groups have focused their efforts on the oncogenic miR-494, as dysregulation of miR-494 might participate in the initial HCC cell dissemination [74]. In addition, high expression of miR-494 in HCC tumor nodules has been linked with poor patient survival [75] (Table 2). Recently, other investigators have proposed the diagnostic and prognostic potential of miRNAs. Jiang et al. reported that a combination of serum levels of miR-10b, mi-106b and miR-181a could be used as an accurate HCC screening panel [76]. Huang et al. developed a circulating miRNA signature to assess the risk of HCC in cirrhotic patients. Using this distinguishable circulating miRNA

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Table 2 Prognostic MicroRNAs for HCC, expression at HCC tissue and clinical application. Name of MicroRNAs

Expression at HCC tissue

Prognostic value

Reference

miR-23a

Upregulated

[73]

miR-494

Upregulated

miR-26

Downregulated

miR-99a, -124, -139, -145 and -199b

Downregulated

miR-222, -135a, -155, -182, -10b and -17-5p miR-21 and -221 miR-10a, -18b, -143, -210, -216a, -224, -301a, -550a and -590-5p miR-18a, -519d and -657 miR-1, -7a, -195, -200a, -203, -214, -219-5p, -376a, -449, -450a and -520e miR-26a/b, -125a/125b, -223 miR-34a, -101, -122, -139

Upregulated Upregulated Upregulated

Higher levels in patients with focal lesion 5 cm or more in size, patients with multiple focal lesions and Okuda stage III Correlated with tumor differentiation, TNM stage and lymph node metastasis Reduced survival but had a favorable response to adjuvant therapy with interferon alpha. Associated with poor prognosis, shorter disease-free survival and features of metastatic tumors Increased risk of tumor recurrence and shorter overall survival Associated to tumor stage and poor prognosis Correlated to metastasis and multiple tumor nodules

Upregulated Downregulated

Correlated to poor differentiation and proliferation Associated to tumor cell proliferation

[117] [117]

Downregulated Downregulated

Correlated to poor survival Metastasis and tumor progression

[117] [117]

[74,75] [120] [114] [114] [116] [117]

Table 3 Circular RNAs, expression at HCC tissue and clinical application. Name of circular RNA

Expression in HCC tissue

Clinical application

Reference

cSMARCA5 circ 0067934 hsa circ 0001727 hsa circ 0001649 hsa circ 0005075 Cdr1as hsa circRNA 104351, hsa circRNA 102814, hsa circRNA 103489, hsa circRNA 102109 and hsa circRNA 100381 hsa circRNA 100327, hsa circRNA 101764, hsa circRNA 101092, hsa circRNA 001225 and hsa circRNA 102904

Downregulated Upregulated Downregulated Downregulated Upregulated Upregulated Upregulated

Diagnosis and prognosis Prognosis Diagnosis and prognosis Prognosis Diagnosis and prognosis Prognosis

[83,84] [86] [85] [87] [118] [121] [119]

profile in combination with conventional clinical predictors, they identified a subgroup of cirrhotic patients with significantly higher risk of developing HCC compared to other patients [77]. HCC miRNAs targeted therapy can be possible by either introducing tumor suppressive miRNAs, inhibiting oncogenic miRNAs or modulating upstream regulators of miRNA expression. Interestingly, the administration of a synthetic RNA molecule developed to mimic the effects of miR122 (agomir-122), reduced the total tumor number and tumor size in a mouse model of liver cancer [70]. A group of researchers reported that targeting HCC cell lines with pre miR-199a-3p oligonucleotides reduced cell proliferation only in CD44+ cell lines. Later they found that CD44 is directly targeted by miR-199a-3p [78]. These results show that miRNAs may be suitable targets for the development of a next generation of HCC treatments (most important miRNAs are summarized in Table 2).

3. Circular RNAs Circular RNAs (circRNAs) belong to the group of non-coding RNA derived from precursor messenger RNAs (pre-mRNA) [79]. First reported several decades ago [80], the circRNAs function has been unknown until recently. Advances on bioinformatics and highthroughput sequencing helped identifying numerous circRNAs in different tissues and stimulating the study of their role in human physiologic and pathologic processes. As a result, circRNAs are now known to have important participation in critical biological processes such as autophagy, cell cycle, apoptosis and proliferation [81]. Several characteristics of circRNAs such as the high degree of conservation across species, cell-type or stage specificity, stability and abundance make this molecules promising as potential new biomarkers.

Downregulated

[119]

circRNAs expression has been linked with several pathological processes such as Alzheimer’s disease, atherosclerosis, diabetes and a variety of cancers, including colorectal, gastric, cervical, breast and HCC. Current available data supports the contribution of circRNAs in the process of tumorigenesis, and in the progression and dissemination of the tumor through the regulation of transcription factors, alternative splicing, and gene expression signatures [81,82]. The regulation of diverse circRNAs have been reported in HCC cell lines. Zhang et al. measured lower plasma cSMARCA5 levels in HCC patients compared to cirrhosis, hepatitis B, and healthy controls. They also evaluated the HCC diagnostic efficiency of plasma cSMARCA5 levels. As previously observed with other types of biomarkers, the combination of cSMARCA5 and AFP levels showed a better efficiency than any of the biomarkers alone to differentiate HCC patients from healthy controls, cirrhotic patients or hepatitis B patients [83]. In a recent study by Yu et al., showed a lower expression of the circRNA derived from the SMARCA5 gene known as cSMARCA5 (hsa circ 0001445), was significantly associated with overall survival and recurrence-free survival in HCC patients after hepatectomy [84]. Inhibition of HCC cells proliferation and migration by cSMARCA5 was also observed in vivo and in vitro experiments [54]. Yao et al. studied the expression of circZKSCAN1 (has circ 0001727) in HCC samples and cell lines. They reported a statistically significant downregulation of circZKSCAN1 in HCC tissues compared to the adjacent non-tumor. Importantly, CircZKSCAN1 expression varied among patients with cirrhosis, different tumor sizes, vascular invasion, microscopic vascular invasion and tumor grade [85]. These data suggest that circZKSCAN1 may represent a suitable biomarker for HCC diagnosis and prognosis.

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High expression of another circRNA named circ 0067934 has been identified in HCC tissues and linked to proliferation, migration and invasion through the inhibition of miR-1324 and activation of the Wnt/␤-catenin signaling pathway [86]. In contrast, the lower expression of hsa circ 0001649 in HCC tissues compared to adjacent non-tumor tissue, significantly correlated with the tumor size and tumor thrombus [87]. Additional important circular RNAs with potential as biomarkers in HCC are summarized in Table 3. 4. Somatic genetic alterations in HCC Genetic mutations have recently gain especial interest due to their possible relation with HCC’s risk factors in addition to their possible use as therapeutic targets. Some of these mutations include CTNNB1, TP53, AXIN1, TERT promoter, ARID1A and ARID2. A recent study demonstrated a correlation between risk factors and particular mutations. More specifically, a relation of CTNNB1 with alcohol and TP53 with HBV was found in HCC patients [88]. Prognostic potential has been given to ARID2 due to its association with liver fibrosis and hepatic vein invasion and progression in liver cancer. Moreover, different stages have been associated with specific alterations, for instance, TERT promoter mutations have been found in early tumor stages whereas TP53 is mutated at late stages [88,89]. Authors have propose diverse genetic mutations as therapeutic targets such as ARID2, which suppresses interferon-␣induced Jak-STAT signaling in HCC patients with HCV infection. 5. Genetic molecular signatures for prognosis and treatment in HCC Genetic profiling has identified and grouped HCCs with similar characteristics, generating a new classification with prognostic relevance. Recent data has shown that different lesions in the same patient could have distinct genetic profiles [90]. The 5-gene score HCC classification describes the G3 subgroup, hepatoblast signature, metastasis signature, Mir26, proliferative subclass/Cluster A [91]. Recently, Nishioka et al. described an association between AFP levels and gene sets with clinical implications. They found a correlation between AFP levels >400 ng/ml and MYC oncogene activation in HCC. Additionally, patients included in this group had worse clinical outcomes [92]. Different groups have found genetic profiles correlated with therapeutic response [93].

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been placed on the CD44v isoforms, which are expressed only under specific scenarios, particularly in advanced stage cancers [97]. CD44 can integrate and transduce multiple micro-environmental cues from growth factors and cytokines to membrane-associated cytoskeletal proteins or to the nucleus. CD44 is involved in the regulation of gene expression related to cell-matrix adhesion, cell migration, proliferation, differentiation and cell survival [97]. In cancer cells is, therefore, a major regulator of the tumor fate. Several studies had described the high expression of CD44 as a poor prognosis predictor in many cancers, including HCC. Our group has recently published that CD133+ /CD44+ lesions associate with poorly differentiated HCC. Remarkably, CD44 or CD133 positivity in these lesions in conjunction with the presence of MVI was strongly associated with worse overall survival and diseasefree survival. Henry et al. published that the presence of high CD44 expression is associated with elevated AFP levels. Other investigators have targeted CD44 using miR-199a-3p to sensitize HCC cells to doxorubicin [78].

6.2. CD133 CD133, also known as human prominin-1, is a transmembrane glycoprotein specifically associated with plasma membrane protrusions in embryonic epithelia. CD133 function may contribute to the organization of plasma membrane topology, and its expression sustains the stem cell phenotype in several malignancies including colon, pancreas, breast, melanoma, lung, brain and prostate. CD133-positive cells demonstrated a significant upregulation of Wnt/␤-catenin, Notch, Hedgehog/SMO, Bmi, and Oct3/4 expression compared to the negative counterparts. These genes are involved in the regulation of important signaling pathways implicated in stem cell proliferation, pluripotency, differentiation and self-renewal. In a mouse model, CD133+ cells were able to initiate tumors that were more aggressive that those generated from CD133− cells [98]. Additionally, our group found that CD133+ was associated with elevated levels of AFP in patients with HCC undergoing liver transplantation. When associated with microvascular invasion (MVI), CD133 independently correlated with poor overall survival (OS) and increased risk of recurrence after transplantation. CD133+ has also been linked to doxorubicin and fluorouracil resistance in HCC.

6. Cancer stem cells markers 6.3. CD90 HCC, like many other types of tumors, contains a heterogeneous population of cancer cells. Studies have revealed a minor subpopulation of HCC tumor cells that holds the ability of self-renewal and differentiation referred as stemness. Due to their resemblance with the stem cells, they are known as cancer stem cells (CSCs) [94,95]. CSCs exhibit great efflux potential, dysregulation of signaling pathways, slow growth and ability to form spherical colonies. These characteristics seem to contribute to their high resistance to chemo and radiotherapy [95,96]. Current approved treatments for cancer are mostly focused on the bulk tumor cells. However, emerging research urges the need to also target the unique properties of CSCs. The identification of specific phenotypic markers for the CSC population in primary HCC tumors may help to advance in the development of new, more effective cancer therapeutics. 6.1. CD44 CD44 is a multistructural and multifunctional transmembrane glycoprotein. It works as a receptor for hyaluronic acid (HA) and as a co-receptor for growth factor and cytokines. Especial attention has

CD90, also known as Thy-1, is a GPI-anchored protein expressed by diverse types of cells. It plays a role in the regulation of apoptosis, cell adhesion, migration, fibrosis, and cell-cell and cell-matrix interactions [99]. All of these processes are considered critical in cancer pathology. In vivo results demonstrated that CD90+ increased during HCC progression [99]. Xenotransplantation of EpCAM+ /CD90+ cells from primary HCCs into immunodeficient mice showed a high metastatic potential by the CD90+ cells in the lung. CD45− CD90+ cells were found in all the specimens of human HCC tissues, but not in normal and cirrhotic liver tissues. Isolation of CD45− CD90+ from blood and subsequent injection into the liver of immunodeficient mice resulted in the development of liver tumors [100]. Clinicopathological analyses revealed an association between CD90+ cells and high incidence of organ metastasis [101]. In addition, the expression of CD90 is also associated with drug resistance in HCC [99]. Overall, these results demonstrate the critical role that CD90 plays on HCC development and progression. Therefore, the direct targeting of CD90 should be further investigated as a potential therapeutic alternative for HCC patients.

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6.4. EpCAM

8.2. Hepatic stellate cells (HSCs)

Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein that plays a functional role in cell–cell homophilic interactions in the mucosal epithelium. Several studies have reported the overexpression of EpCAM in diverse types of cancer. EpCAM+ cells from HCC tumor specimens showed stem cell traits such as self-renewal and differentiation features [102]. The tumorigenic potential of EpCAM+ cells has been demonstrated by the ability to form tumors in immuno-deficient mice [103]. Treating HCC cells with the GSK-3␤ inhibitor BIO, which activates the Wnt/␤-catenin signaling pathway, promotes the increase EpCAM+ cells, suggesting that EpCAM expression is regulated by the Wnt/␤-catenin pathway [103]. EpCAM+ proliferating ductal cells (PDCs) originate HCC in injured liver. Interestingly the Wnt/␤catenin pathway was upregulated in the PDC-derived tumors, which presented a concomitant cholangiocellular carcinoma component [104]. Using RNA interference to block EpCAM expression resulted in inhibition of spheroid formation, decreased cellular invasion and tumorigenicity of HCC cells [103]. These results illustrate the therapeutic potential of EpCAM as molecular target.

Also known as Ito cells, HSCs are the principal responsible for fibrosis or synthesis of collagen in the liver. As a consequence of repetitive liver injury, HSCs constantly differentiate into myofibroblasts-like cells, which are more contractile. These actively secreting cells are responsible for the synthesis of extracellular matrix components [105]. HSCs are not only responsible for fibrosis, but they have been also implicated in the proliferation and dissemination of HCC cells [107] through the secretion of angiogenic factors like VEGF, angiopoietin 1 and 2. In addition, Xu et al. revealed the role of HSCs in the progression of HCC by enhancing immunosuppressive cell populations such as myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) mediated by the activation of the COX2-PGE2-EP4 pathway [108].

7. Tumor stroma/tumor microenvironment Cancer research studies performed in vivo in animal models have determined the importance of stromal cells in the tumorigenesis and progression of the tumor. Stromal cells are essential to maintain the architecture and homeostasis of normal tissue, and also of cancer [105]. Stromal cells are part of the tumor microenvironment (TME) that surrounds the tumor and provides the necessary conditions for its growth. The crosstalk between tumor cells and the TME plays a critical role on the regulation of the tumor initiation, progression and metastases and may determine the tumor cell fate. The main components of the TME are fibroblasts, myofibroblasts, immune cells, inflammatory cells, and the extracellular matrix (ECM), which are classically divided on two main groups, the cellular and the non-cellular components.

8. Cellular components 8.1. Cancer associated fibroblasts (CAFs) CAFs is a subpopulation of fibroblasts found in the TME and involved in the modulation of cancer [105]. CAFs are known to be a major player in tumor formation, progression and dissemination. Since the majority of HCC originates from primary fibrous liver injury, it seems clear that fibroblast related cells are tightly involved in the pathogenesis of HCC. Because of their great content of Rough Endoplasmic Reticulum and Golgi apparatus, CAFs are highly active in the synthesis and secretion of proteins. A group of proteolytic enzymes called matrixmetalloproteinases (MMP) are between these proteins. Specifically MMP-2, MMP-3 and MMP-9 have been identified as produced by CAFs. The proteolytic ability of these enzymes is involved in different physio-pathological processes, including inflammation and tumor invasion [17]. CAFs can also secrete cytokines to gather inflammatory and endothelial cells involved in the tumor genesis and maintenance. Sukowat et al. demonstrated that HCC cells cultured with CAFs upregulated the gene expression of TGF␤1 and fibroblast activated protein, which may induce a switch from normal tissue to TME [106].

8.3. Tumor-associated macrophages (TAMs) One of the most important and well-studied component of the TME are the macrophages. These TAMs are also known as Kupffer cells in the liver, and are involved in phagocytosis and tissue remodeling. TAMs can secrete numerous factors that can affect both TAM themselves and the tumor. Macrophages can be fully polarized into two specific phenotypes M1 and M2. M1 are induced by the Th1 cytokine, IFN-␥ or by microbial antigens such as Lipopolysaccharides (LPSs) and their main function is to phagocyte microbes, produce pro-inflammatory cytokines and initiate an immune response. The other subtype is M2, which are alternatively induced by Th2 cytokines IL-4and IL-13, or glucocorticoids. Their main role is to decrease inflammation and promote tissue repair. It has been stablished that M1s are replaced by M2s in later stages of HCC. Since M1 macrophages try to eliminate tumor cells, the substitution by M2 is linked with is the tumor proliferation and ECM remodeling [105]. In addition, M2 macrophages can secrete angiogenic factors such as VEGF and EGF, which contribute to tumor expansion [17]. A study demonstrated a negative correlation between M2 CD206-positive macrophages and overall survival in HCC [109]. 8.4. Lymphocytes A tumor cell expresses antigens that are not found on normal counterparts. These antigens, called tumor-associated antigens (TAAs), are recognized by the adaptive immune system to elicit a protective anti-tumor response. However, tumors develop several mechanisms to evade immune surveillance. The level of tumorinfiltrating lymphocytes (TILs) has been significantly correlated with HCC prognosis. Several studies demonstrated a worse prognosis in HCC patients based on a ratio between anti-tumor effector T cells and suppressor Tregs in the tumor [107]. A meta- metaanalysis of published studies on the clinical importance of TILs in HCC suggested the use of the effector/regulatory T cell ratio as a prognostic biomarker in HCC. The results showed that high levels of CD3+/CD8+ TILs is associated with better overall survival [110], and high levels of Tregs and low levels of effector T cells with poor outcomes in HCC patients. 9. Non-cellular components 9.1. Transforming growth factor-beta (TGF-ˇ), fibroblast growth factor (FGF), hepatocyte growth factor/scatter factor (HGF/SF) and vascular endothelial growth factor (VEGF) TGF-␤ is a superfamily of cytokines involved in the regulation of cell growth, differentiation, fibrogenesis, angiogenesis, immunosuppression and proliferation in cancer. There has been a strong

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association with fibrosis due to its ability to enhance the differentiation of CAFs. Many studies have demonstrated higher levels of TGF-␤ mRNA in patients with HCC [111]. In diseases with extensive liver fibrosis, the levels of TGF-␤ are up-regulated, inducing the proliferation and activation of HSCs, leading to fibrogenesis. Furthermore, recent reports indicate that TGF-␤ plays a direct role in the genesis of HCC due to a modification from its role as tumor suppressor to an oncogenic factor. In addition, TGF-␤ is capable of activating PDGF, which can intensify the EMT process. Finally, the expression of miR-181b is increased by TGF-␤1 exposure in the hepatocytes resulting in HCC growth, invasion, migration and impact on survival [107]. Yang et al. demonstrated the importance of TGF-␤ as a target therapy in HCC [112]. Fibroblast growth factor (FGF) is a polypeptide cytokine, with important effects on the endothelium. High expression of FGF-19 is strongly correlated to poor prognosis in HCC [107]. In addition, Gao et al. reported that FGF-19 plays a major role HCC resistance to sorafenib, currently the only FDA-approved drug for HCC. Importantly, the inhibition of FGF19/FGFR4 signaling pathway helps overcome the resistance to sorafenib in HCC [113]. HGF, is a cytokine with a critical role in many liver functions including liver regeneration. It has been involved in hepatocarcinogenesis through its association with the c-Met receptor. HGF has been associated to fibrogenesis through interactions with HSCs. In addition, HGF levels higher than 1.0 ng/ml have been associated to poor survival in HCC patients [114]. VEGF, a potent angiogenic factor, has been linked to stimulation of proliferation and migration of endothelial cells. Other effect of VEGF is the increased in vascular permeability, which is one of the steps involved in the development of metastasis. Several authors have suggested a relation between VEGF and HCC metastases. A recent study observed a positive correlation between high levels of VEGF and tumor size, venous infiltration and metastasis, identifying VEGF as a risk factor in HCC [115]. VEGF is one of the targets of sorafenib, the drug currently used to treat advanced HCC. 9.2. Summary In conclusion, several biomarkers have been used for diagnosis and prognosis of HCC. AFP, the most commonly used biomarker in liver cancer, has limited value since is not present in 30% of the cases, especially in early stages. Many efforts have been performed to find molecules to improve detection and prognostic accuracy in HCC. Importantly, most recent publications suggest that the combination of 2 or more biomarkers alone and additional demographic information, can improve their sensitivity, specificity and predictive value. Further research is needed to assess novel biomarkers and their clinical implications in patients with HCC. Conflict of interest None declared. References [1] Song P, Tang Q, Feng X, Tang W. Biomarkers: evaluation of clinical utility in surveillance and early diagnosis for hepatocellular carcinoma. Scand J Clin Lab Invest Supl 2016;245:S70–6. [2] Tsuchiya N, Sawada Y, Endo I, Saito K, Uemura Y, Nakatsura T. Biomarkers for the early diagnosis of hepatocellular carcinoma. World J Gastroenterol 2015;21(37):10573–83. [3] Feng M, Ho M. Glypican-3 antibodies: a new therapeutic target for liver cancer. FEBS Lett 2014;588(2):377–82. [4] Bruix J, Boix L, Sala M, Llovet JM. Focus on hepatocellular carcinoma. Cancer Cell 2004;5(3):215–9. [5] Sanyal A, Poklepovic A, Moyneur E, Barghout V. Population-based risk factors and resource utilization for HCC: US perspective. Curr Med Res Opin 2010;26(9):2183–91.

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Please cite this article in press as: De Stefano F, et al. Novel biomarkers in hepatocellular carcinoma. Dig Liver Dis (2018), https://doi.org/10.1016/j.dld.2018.08.019