Molecular and serum markers in hepatocellular carcinoma: Predictive tools for prognosis and recurrence

C

12

3

4

5

1d

Critical Reviews in Oncology/Hematology 82 (2012) 116–140

Molecular and serum markers in hepatocellular carcinoma: Predictivetools for prognosis and recurrence

Ashish Singhal a, Muralidharan Jayaraman b, Danny N. Dhanasekaran b, Vivek Kohli a,∗a Nazih Zuhdi Transplant Institute, INTEGRIS Baptist Medical Center, 3300 NW Expressway, Oklahoma City, OK 73112, USA

b OU Cancer Institute, Oklahoma University Health Sciences Center, 975, NE 10th Street, Oklahoma City, OK 73104, USA

Accepted 18 May 2011

ontents

. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

. Cellular markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1182.1. Proliferating activity of HCC cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1182.2. Nuclear morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192.3. p53 and related genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1192.4. Genomic instability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

2.4.1. Chromosomal instability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1202.4.2. Loss of heterozygosity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1202.4.3. Microsatellite instability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1202.4.4. DNA aneuploidy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

2.5. Cell cycle regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1212.5.1. Cyclin and other kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1212.5.2. Cyclin-dependent kinase inhibitors (CDKIs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

2.6. Tumor promoter genes and their receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1212.7. Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1212.8. Telomerase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122

. Cell adhesion and degradation of extracellular matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1223.1. Adhesion molecules and related markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1223.2. Degradation of extracellular matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

. Angiogenesis associated markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1244.1. Microvessel density (MVD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1244.2. Vascular endothelial growth factor (VEGF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1244.3. Platelet-derived endothelial cell growth factor (PD-EGF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1244.4. Hypoxia-inducible factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1244.5. Nitric oxide synthase (NOS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
4.6. Basic fibroblast growth factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1254.7. Tissue factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1254.8. Endostatin/collagen XVIII . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 4.9. Interleukin-8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1254.10. Angiopoietins (Ang-1 and Ang-2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
. Growth factors and their receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1255.1. Transforming growth factor-beta (TGF-�s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1255.2. Tumor-specific growth factor (TSGF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

∗ Corresponding author.E-mail address: [email protected] (V. Kohli).

040-8428/$ – see front matter © 2011 Published by Elsevier Ireland Ltd.oi:10.1016/j.critrevonc.2011.05.005

dx.doi.org/10.1016/j.critrevonc.2011.05.005

mailto:[email protected]

dx.doi.org/10.1016/j.critrevonc.2011.05.005

6

7

8

91

A

iipppt©

KS

1

ceruvcoihdd(

cctr

A. Singhal et al. / Critical Reviews in Oncology/Hematology 82 (2012) 116–140 117

5.3. Epidermal growth factor receptor family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265.4. Hepatocyte growth factor/scatter factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

. Oncofetal and glycoprotein antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1266.1. Alpha-fetoprotein and alpha-fetoprotein-L3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1266.2. Glypican-3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

. Enzymes and isoenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1277.1. Gamma-glutamyl transferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1277.2. Alpha-l-fucosidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1277.3. Des-gamma-carboxyprothrombin (DCP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1277.4. Golgi phosphoprotein 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

. Genetic biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1288.1. Alpha-fetoprotein mRNA (AFP mRNA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1288.2. Gamma-glutamyl transferase mRNA (GGT mRNA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1288.3. Insulin-like growth factor II-mRNA (IGF-II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1288.4. Albumin mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1288.5. Human telomerase reverse transcriptase mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1288.6. MicroRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1300. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

bstract

With increased understanding of cancer biology, a multitude of pathological, genetic, and molecular events that drive hepatocarcinogenesis,ncluding angiogenesis, invasion, and metastasis, has been identified. Lately, they are being aggressively evaluated due to challenges involvedn establishing early diagnosis, optimizing therapy for cancer inducing hepatotrophic viruses, minimizing the emergence of new tumors, andreventing recurrence after surgical resection or liver transplantation. This comprehensive review examines and critiques the evidence fromublished manuscripts reporting various tissue and serum biomarkers involved in hepatocellular carcinoma. These markers not only help inrediction of prognosis or recurrence, but may also assist in deciding appropriate modality of therapy and represent novel targets for potentialherapeutic agents.

2011 Published by Elsevier Ireland Ltd.

eywords: Markers; Molecular; Genetic; Prognosis; miRNA; HCC; Hepatocellular; Carcinoma; Liver; Cancer; Recurrence; Survival; Biomarkers; Tissue;

trtrspwtaTimEeShn

erum

. Introduction

Hepatocellular carcinoma (HCC) represents the fifth mostommon cancer affecting approximately one million peoplevery year worldwide, with an incidence equal to the deathate [1,2]. In more than 80% of patients, HCC arises in thenfavorable setting of cirrhosis, most often related to chroniciral hepatitis B or C [3]. Other important causes includehronic viral hepatitis B (HBV) plus D viruses, alcohol abuse,besity, hemochromatosis, �1-antitripsin deficiency, and tox-ns similar to aflatoxin [1–3]. Although, the prevalence isighest in Southeast Asia and sub-Saharan Africa, the inci-ence is rising in Europe and United States over the last twoecades, mainly due to increasing rates of hepatitis C virusHCV) infection and alcoholic liver disease [2,4,5].

Surgery including liver transplantation remains the onlyurative modality for HCC. However, despite resection with
urative intent, the clinical course is variable and the longerm prognosis is poor with reported 5-year survival ratesanging from 17% to 53%. The recurrence rates after ortho-
t

d

opic liver transplantation (OLT) within Milan criteria alsoemained high ranging from 10 to 20% with in the first yearo the cumulative 5 year recurrence of 75–100% [6–8]. Theseecurrence rates do not reflect simply size and number asuggested in the initial Milan series, but a complex inter-lay of unknown host and tumor related factors associatedith aggressive tumor biology. Attempts have been made

o predict recurrence and prognosis in patients with HCCfter hepatectomy using clinicopathological characteristics.hese include tumor size, associated cirrhosis, HBV or HCV

nfection, presence of daughter nodules, adequate resectionargin, vascular permeation, absence of capsule, histologicaldmondson grade of tumor differentiation [9–12]. How-ver, they lack sensitivity for predicting individual prognosis.erum tumor markers, particularly alpha-fetoprotein (AFP)as been found to be prognostic; however, they rely on sig-ificant tumor burden making their usefulness in operable
umors questionable.
With advances in understanding of tumor biology andevelopment in cellular and molecular techniques, interests

118 A. Singhal et al. / Critical Reviews in Oncology/Hematology 82 (2012) 116–140

Table 1Various markers involved in hepatocarcinogenesis.

1 Proliferating indices PCNA-LI, KI-67-LI, MIB-1, CAS, nm-23, MAD22 Nuclear morphology Nuclear area3 p53 and related genes p53, MDM2, p73, NPRL24 Genomic instability CSI, LOH, MSI, aneuploidy5 Cell cycle regulators cyclin-a, cyclin-D, cyclin-E, Aurora kinase B, CDKIs, p276 Tumor promotor genes ras, c-myc, c-fms, TOP2A, ets-1, Alpha B-crystallin7 Apoptosis Bax, Bak, Bcl-xS, Fas, Survivin8 Replicative potential Telomerase (including TERT)9 Tissue invasion and metastasis E-cadherin, Wnt/�catenin, osteopontin, CD44 isoforms, sICAM-1, RhoC-GTPase, deleted in liver cancer 2

(DLC2), MMP’s, uPA, PAI-110 Angiogenesis related MVD, VEGF, PD-EGF, HIF, NOS, b-FGF, IL-8, angiopoeitins11 Growth factors TGF-�, TSGF, leptin, EGFR,12 Genetic biomarkers AFP mRNA, GGT mRNA, IGF-II, Albumin mRNA, hTERT mRNA13 MicroRNAs miR15b, miR18a, miR21, miR23b, miR26a, miR29, miR34a, miR101, miR122, miR125b, miR143, miR195,

miR221, miR223, miR224, miR338, miR378, miR500

PCNA-LI: proliferating cell nuclear antigen labeling index, MIB-1: mindbomb homolog 1, CAS: Chromosome Segregation gene homolog, nm-23: non-metastatic protein-23, MAD2: mitotic arrest defective protein 2, CSI: chromosomal instability, LOH: loss of heterozygosity, MSI: microsatellite instability,CDKIs: cyclin dependent kinase inhibitors, TOP2A: topoisomerase II alpha, TERT: telomerase reverse transcriptase, DLC2: deleted in liver cancer 2, MMP:matrix metalloproteases, uPA: urokinase plasminogen activator, MVD: microvessel density, VEGF: vascular endothelial growth factor, PD-EGF: platelet-derivedendothelial growth factor, HIF-1�: hypoxia-inducible factor-1 alpha, NOS: nitric oxide synthase, b-FGF: basic fibroblast growth factor, IL-8: interleukin-8,T eptor, TG th fact

hiamriauhntpogiiamaod

2

2

(1Aoni

lkDieptocihoiHarLs

airsaaloai[s

GF-�: transforming growth factor beta, EGFR: epidermal growth factor recGT mRNA: gamma-glutamyl transferase mRNA, IGF-II: insulin-like grow

ave grown in the role of molecular markers in biolog-cal behavior, prognostic significance, and their potentials therapeutic targets. A large number of tissue and serumarkers (Table 1) associated with invasiveness, metastasis,

ecurrence, and potential prognostic significance have beendentified [13–16]; however, specific markers and their reli-bility are currently lacking. In this article, we present anp to date review on tissue and serum markers involved inepatocellular carcinoma, and discuss their role in prog-osis and prediction of recurrences. Prognostic value ofhe following molecular markers are discussed: tumor sup-ressor and promoter genes, telomerase activity, markersf genomic instability, proliferating activity of tumor cells,enes regulating DNA damage, cell cycle regulators, genesnvolved in growth inhibition and apoptosis, markers ofnvasion and metastasis-genes responsible for cell–cell inter-ction proteinases that degrade extracellular matrix, adhesionolecules, angiogenic factors, growth factors and receptors,

nd microRNAs (miR). In addition, serum markers includingncofetal antigens, enzymes, and inflammatory markers areiscussed in detail.

. Cellular markers

.1. Proliferating activity of HCC cells

Many antigens such as proliferating cell nuclear antigenPCNA), Ki-67, Mcm2, mind bomb homolog (MIB-), MIA, and Chromosome Segregation (CSE1L)/Cellular
poptosis-Susceptibility (CAS) have been used as markersf proliferation activity for cancer cells [17]. PCNA was origi-ally identified as a 36 kDa nuclear antigen whose expressions often associated with proliferating and oncogenic cellu-
toa

SGF: tumor specific growth Factor, AFP mRNA: alpha-fetoprotein mRNA,or-II, CRP: c-reactive protein, miR: microRNA.

ar phenotypes [18]. PCNA has been identified to play aey role in DNA replication through its interaction withNA polymerase-ä and -e and it has been observed to show

ncreased expression in highly proliferating cells. Thus, thexpression of PCNA has been used as a proliferation andrognostic marker in a variety of tumors [19]. Consistent withhese observations, immunohistochemical (IHC) detectionf PCNA over-expression combined with histopathologicalharacteristics, the PCNA labeling index (PCNA-LI) wasdentified as marker for HCC [20,21]. Subsequent studiesave successfully focused on using the increased expressionf PCNA expression in evaluating malignant grade, predict-ng recurrence time and prognosis in HCC patients [22].igh PCNA-LI was found in large tumors without a capsule

nd associated with more aggressive growth and recurrence,esulting in low survival rates [23]. Thus, the use of PCNA-I as a prognostic predictor for HCC has been established ineveral clinical studies [23–25].

Ki-67 is another nuclear antigen, which is widely useds a proliferation-specific marker, since it is only presentn proliferating cells. Ki-67, a putative factor involved inRNA transcription, is an absolute requirement for progres-ion through the cell-division cycle I [26]. Its presence duringll active phases of the cell cycle G1, S, G1–M phases,nd absence in resting cells (G0 phase) makes it an excel-ent marker for determining the so-called growth fractionf a given cell population [27]. In HCC, Ki-67 expressionnd the fraction of Ki-67-positive tumor cells (Ki-67 label-ng index) is often correlated with the tumor growth rate16,17]. Higher Ki-67 labeling index (Ki-67-LI) has a veryimilar clinical significance to PCNA-LI in HCC, reflecting
he existence of biologically aggressive phenotypes and poorverall and disease-free survival rates [16,23,28,29]. MIB1,monoclonal antibody raised against Ki-67, thus known as

Oncolo

MKtTpct

aghtpCaamioicCppRCrtiar

2

titnciwpalnnc(alrpptq

(s(nc

2

ooilitrbfcodsahocDfm

0dstaiaipeSpta

car7wms

A. Singhal et al. / Critical Reviews in

IB1-index, is used to evaluate the expression levels ofi-67. MIB-1 index can be easily assessed by simple IHC

echnique on histological tissues including liver biopsies.umors were scored as low proliferating (<10% positive neo-lastic cells) or high proliferating (>10% positive neoplasticells) based on the ratio of MIB-1 positive nuclei with respecto the total number of neoplastic cells [30].

In addition to PCNA and Ki-67, a nuclear protein knowns Cellular Apoptosis-Susceptibility (CAS) was also investi-ated as a prognostic predictor for HCC. CAS is a humanomolog of Chromosome Segregation or CSE1L appearso be involved in ran-mediated nuclear transport of criticalroteins involved in cell proliferation and apoptosis [31].AS expression is generally upregulated in many cancersnd depletion of CAS is correlated with cell death [32]. Asnuclear transport factor, CAS/CSE1L plays a role in theitotic spindle checkpoint assuring genomic stability dur-

ng cell division, which is frequently disturbed in neoplasiasf various origins, including HCC [33]. Based on the find-ngs that a stronger expression of CAS could be seen in liverells committed to proliferation, and the recent findings thatAS is expressed at higher levels in human HCC tissues com-ared to normal tissues, it has been suggested that CAS couldrove to be a predictive prognosis marker for HCC [33,34].ecently, it has been shown that the ectopic expression ofAS enhances invasion and metastasis of melanoma cells

ather than their proliferation [35]. This observation takenogether with the previous findings of CAS overexpressionn cancer tissues, it has been concluded that CAS can be useds a marker for tumor metastasis. Such a critical correlationemains to be established for HCCs.

.2. Nuclear morphology

Nuclear profiles have been reported as useful prognos-ic predictors in various cancers, including HCC. In a studynvolving 76 patients with HCC who underwent hepatec-omy, a correlation was evaluated between the morphologicaluclear features (nuclear area, perimeter, and shape) andlinicopathological parameters using the computer-assistedmage analysis system. Morphometric data were comparedith the patient survival, clinicopathological status, and theroliferative activity of cancer cells [36]. The mean nuclearrea of poorly differentiated carcinoma was significantlyarger than that of moderately and well differentiated carci-oma and a significant correlation was detected between theuclear area of cancer cells and proliferative activity asso-iated with proliferating cell nuclear antigen labeling indexPCNA-LI) of cancer cells. Vascular invasion or intrahep-tic metastasis was more frequently detected in patients witharge nuclear areas (>50 microm2) and the 5-year survivalate of these patients was significantly lower than that of the

2
atients with small nuclear areas (<50 microm ) [37]. Com-uterized nuclear morphometry is more objective and quickerhan conventional microscopic analysis [36]. Recently, auantitative assessment of nuclear morphometry descriptors
wtam

gy/Hematology 82 (2012) 116–140 119

NMD) of cancer cells by computer-assisted image analy-is has been used to formulate the quantitative nuclear gradeQNG) system to predict the clinical, diagnostic, and prog-ostic outcome in cancer patients which may reduces theomplexity of patient disease management [37].

.3. p53 and related genes

p53 gene is an established tumor suppressor gene locatedn chromosome 17p13.1 and is responsible for regulationf the cell cycle at G1/S and G2/M interfaces, as well asnduction of apoptosis in response to severe damage to cellu-ar DNA. p53 dysfunction can induce abnormal cell growth,ncreased cell survival, genetic instability, and drug resis-ance. Mutations in the p53 gene are the most frequentlyeported somatic gene alteration in human cancer and haveeen associated with higher grade and more advanced stageor cancers of various origins. In addition, p53 mutation isonsidered as a strong marker predicting an increased riskf local relapse, treatment failure, and shorter overall andisease-free survival in many kinds of human carcinomas,uch as breast [38], colorectal [39], esophageal [40], headnd neck [41], lung [42], and ovarian [43]. Several studiesave shown a relationship between the nuclear accumulationf p53 protein and poor disease-free and overall survival ofancer patients. p53 mutations may be detected in plasmaNA of cancer patients, and may be used as a prognostic

actor and an early marker to indicate recurrence or distantetastasis [22].Earlier studies reported p53 mutation with an incidence of

–70% in HCC patients [24,44]; however, Sheen et al. haveemonstrated the mutant p53 gene in >80% of hepatectomypecimens [45]. The wide variation in positivity may be dueo different adopted thresholds of positivity, use of differentnti-p53 antibodies, geographical variations, and differencesn the molecular mechanisms of hepatocarcinogenesis suchs aflatoxin exposure. Studies showed that p53 mutation wasnvolved in dedifferentiation, proliferating activity, tumorrogression, and invasiveness; and may influence the postop-rative course (particularly the recurrence in 1 year) [45–47].troescu et al. showed expression of p53 mutation in a higherercentage (85.7 vs. 42.1%) of undifferentiated histologicalumor grades (Edmondson Steiner G3/G4 vs. G1/G2) [23],nd was significantly related to vascular permeation [45].

Studies assessing the predictive role of immunohisto-hemical detection of p53 expression, or the serum anti-p53ntibodies in prognosis of HCC have yielded conflictingesults [48,49]. In a study involving 79 patients of HCC,6.6% patients with mutation develop tumor recurrenceithin short interval as compare to 40% patients withoututation [45]. In a recent prospective study, p53 overexpres-

ion was found to be the most significant factor associated
ith the overall survival rates of HCC patients after resec-
ion and its significance was even greater than factors suchs tumor size, vascular invasion, and tumor capsule. Further-ore, the 3-year and 5-year overall survival rates of HCC

1 Oncolo

pleftamavrbctr

cpbppppitsup

2

2

mieSh1I1Katpe1panrmgplar

gbtvalhtfcomtdttf

2

fmassCLtwcatgpts

2

DoBrimutawMH(

20 A. Singhal et al. / Critical Reviews in

atients with positive p53 nuclear accumulation were muchower than those of the HCC patients with negative p53xpression [49]. Serum anti-p53 antibody could also be a use-ul prognostic factor for HCC patients [48]. p53 mutation inhe primary lesion is an useful independent prognostic factorffecting survival after recurrence [22,50]. Post recurrenceedian survival in p53 mutation positive patients were lower

nd suggested immunohistochemical detection of p53 as aaluable tool for prediction of recurrence in patients afteresection or for identifying subgroups of patients who maye at higher risk [23]. Lowe et al. suggested that p53 statusould also be an important determinant of tumor response toherapy as few point mutations on p53 may produce treatmentesistant tumors [51].

MDM2 protein is overexpressed in several types of can-ers. The transcription of the MDM2 gene is activated by53 and this limits the growth-suppressing activity of p53y direct binding. In a study involving 107 patients, MDM2ositivity in the nuclei of HCC cells was detected in 26%atients and the expression of MDM2 showed a significantlyositive correlation with expression of p53 mutation [52]. p73rotein, a first identified homolog of p53 gene, is known tonduce apoptosis. A study involving 193 patients with cura-ively (R0) resected HCC showed p73 expression in 32%pecimens. p73-positive tumors had a poorer prognosis onnivariate survival analysis and identified as an independentrognostic factor on multivariate Cox survival analysis [53].

.4. Genomic instability

.4.1. Chromosomal instabilityChromosomal instability (CIN) is the loss or gain of chro-

osomal segment during cell division and leads to an increasen aneuploidy, which in turn cause further mutations andnhance tumor progression leading to aggressive tumors.imilar to other human malignancies, HCC demonstrates aigh incidence of CIN. Katoh and colleagues found loss on7q13.3 and gain on 8q11 as independent prognostic factors.n addition, chromosomal losses on 4q, 8p, 13q, 14q, and7p were found to be associated with shorter survival [54].usano et al. correlated the total number of chromosomal

lterations with increasing tumor stage and suggested thatotal number of chromosomal alterations were independentlyrognostic on multivariate analysis. A gain of 8q24 was pref-rentially observed in well differentiated HCCs and a loss of3q13–14 and amplification of 11q13 were associated withoorly differentiated tumors. Furthermore, loss of 8p and 13qnd amplification of 11q13 were associated with poor prog-osis and increased risk of recurrence [55]. Itano et al. usedestriction landmark genomic scanning (RLGS), a screeningethod for multiple genomic changes, to detect unknown

enetic alterations in HCC. Disease-free survival rates for
atients with >16 changed RLGS spots were significantlyower than patients with RLGS spots [56]. In multivari-te analysis, the number of changed spots was proven toetain an independent prognostic value. These results sug-
aa(

gy/Hematology 82 (2012) 116–140

est that the number of changed RLGS spots may be a usefuliological marker for recurrence of HCC. Another impor-ant area that should be paid attention to is the prognosticalue of circulating DNA in plasma or serum, and its geneticlterations in cancer patients. Small amounts of DNA circu-ate in both healthy and diseased human plasma and serum;owever, plasma of cancer patients had increased concen-rations of DNA. Characteristics of tumor DNA have beenound in genetic material extracted from the plasma of can-er patients including decreased strand stability, presencef specific oncogene or tumor suppressor gene mutations,icrosatellite alterations, Ig rearrangements, and hyperme-

hylation of several genes. These results obtained in manyifferent cancers have opened a new research area indicatinghat plasma DNA might eventually be a suitable target forhe development of noninvasive diagnostic, prognostic, andollow-up test for cancer.

.4.2. Loss of heterozygosityKnudson described loss of tumor suppressor gene as a

requent event in liver tumorogenesis and suggested thatalignant transformation involves a constellation of gene

lterations occurring in a temporal fashion [57]. Loss of tumoruppressor gene was based on loss of heterozygosity (LOH)ituated within or adjacent to specific genes of interest (APC,DKN2A, DCC, MET, MYC1, OGG1, p34, p53, PTEN).OH can be detected in plasma DNA and could be of prognos-

ic significance in HCC patients [58]. LOH in plasma DNAas more frequently detected in the patients with intrahepatic

ancer metastasis. Marsh et al. suggested that the measure ofllelic loss of heterozygosity combined with tumor number,umor size, vascular invasion, lobar distribution, and patientender provide a highly discriminatory model compared toathological Tumor-Node-Metastasis (pTNM) staging sys-em for predicting cancer recurrence and recurrence-freeurvival after liver transplantation [58].

.4.3. Microsatellite instabilityMicrosatellite instability (MSI) is caused be mutations in

NA mismatch repair genes. Studies have reported MSI atne chromosomal locus ranging from 0% to 68% [59,60].AT26, a microsatellite marker that reliably predicts high

ate-MSI (MSI-H: defined as MSI in >30% of markers exam-ned) was found to be rarely altered [60]. These differencesight be due to different etiologies, different approaches

sed to define the presence of microsatellite mutator pheno-ype, differences in the type and number of markers analyzed,nd differences in the cut-off criteria. In a study, 37 patientsith HCC affecting non-cirrhotic livers were analyzed forSI according to the criteria defined for colorectal cancer.igh MSI (MSI-H > 30%) was found in 16% and low MSI

MSI-L < 30%) in 27% of HCCs. MSI-H was significantly
ssociated with more aggressive histological tumor featuresnd a shorter recurrence-free survival than those without17% vs. 47%, at 36 months) [61].

Oncolo

2

wbvtGusws

2

2

ughistmessaelsp

2

iItcc

pgcWc[acam[tepyt

giswosf1peoasagtpve

2

mptwtrpca1fremvhtnstwas

2

o


.4.4. DNA aneuploidyMany reports indicate that aneuploidy is common in HCC

ith reported rates varying between 35% and 84% and coulde a predictive marker for HCC prognosis. The overall sur-ival rate of patients with aneuploid cells is much lower thanhat of patients with diploid ones and those with multiple0/G1 peaks have the worst prognosis [16]. Tumor ane-ploidy was found to be associated with reduced overallurvival following resection and on subanalysis, aneuploidyas found to independently associate with reduced overall

urvival in tumors < 5 cm in size.

.5. Cell cycle regulators

.5.1. Cyclin and other kinasesDisruption of the G1/S and G2/M transition leads to

ncontrolled cell growth, resulting in development and pro-ression of cancers. Overexpression of cyclin A, D1, and Eas been found to correlate with the relapse of HCC and arendependent predictive markers for recurrence and progno-is [62,63]. Chao and colleagues found cyclin A expressiono independently predicting the decreased free survival on

ultivariate analysis [62]. Two studies have found cyclin D1xpression to be associated with decreased free and overallurvival following resection [22,64]. The enhanced expres-ion of cyclin E correlates with hyperphosphorylation of Rbnd a high frequency of Ki-67-positive cells. HCCs withnhanced cyclin E expression probably contain a relativelyarge number of proliferating cancer cells [62]; however, notudies have found cyclin E to be significantly associated withrognosis.

.5.2. Cyclin-dependent kinase inhibitors (CDKIs)CDKIs are potent negative cell cycle regulators inhibit-

ng the G1/S transition. Two families of CDKIs exist, theNK4 family containing p15, p16, p18, and p19 inhibiting D-ype cyclin-CD4/CDK6 complexes, and the KIP/CIP familyontaining p21, p27, and p57, binding and inhibiting variousyclin/CDK complexes.

Methylation of promoter regions of p15 and p16 in HCCatients leads to gene silencing. Li et al. reported that p16ene methylation was only associated with viral hepato-arcinogenesis (hepatitis B virus or hepatitis C virus) [65].eihrauch et al. reported that K-ras mutation may be asso-

iated with p16 inactivation in chemically induced HCC66]. Since many studies found rare homozygous deletionsnd infrequent mutations, Matsuda suggested that epigenetichanges may be the main reason for p16 inactivation in hep-titis virus-associated HCC, while genetic mutations of p16ay occur in specific geographical conditions and pedigrees

67]. Patients with p15 and p16 methylation were more likelyo develop recurrent disease following resection [68]; how-
ver, Matsuda et al. reported that p16 positivity independentlyredicts the increased overall survival on multivariate anal-sis [67]. Loss of p18 expression has also shown to predicthe overall survival on multivariate analysis [69].
BafH

gy/Hematology 82 (2012) 116–140 121

Loss of p27 cooperates with mutations in several onco-enes and tumor suppressor genes to facilitate tumor growth,ndicating that p27 may be a “nodal point” for tumor suppres-ion. Many studies have found high p27 expression in patientsith HCC to be associated with improved disease free andverall survival [62,70]. Ito et al. reported that the p27-LI wasignificantly decreased in cases with portal invasion, poor dif-erentiation, larger size, and intrahepatic metastasis among04 HCC cases, and suggested that p27 can act as an inde-endent predictor of HCC recurrence [71]. Fiorentino et al.xamined 54 HCC cases and reported that high expressionf p27 was a favorable independent prognostic parameternd recurrence occurred in low p27 expressers [70]. In aeries of curatively resected HCCs, p27 was decreased only indvanced cases [72]. In HCC, epigenetic changes of the p16ene were suggested as the main cause for the p27 inactiva-ion. HCCs expressing low levels of p27 (low-p27 expressers;27 LI of <50% in the tumor cells) showed significantly unfa-orable prognoses compared with HCCs showing high p27xpression (high p27 expressers; p27 LI of >50%) [67].

.6. Tumor promoter genes and their receptors

Aberrations of many tumor promoter genes such as ras, c-yc, and c-fms have been indicated as indicators of malignant

otential and poor prognosis in HCC [22]. c-myc amplifica-ion causes the progression of HCC and found to be associatedith significantly shorter disease-free survival [73]. Among

he erb-B receptor family members, c-erbB-2 (Her-2/neu)epresents a well-established prognostic marker and thera-eutic target in several human tumor types, especially breastancer [22]. Topoisomerase II alpha (TOP2A) gene is locateddjacent to the Her-2 oncogene at the chromosome location7q12–q21 and is either amplified or deleted, with equalrequency in Her-2 amplified tumors. By tissue microar-ay analysis of 172 HCC patients, a study found TOP2Axpressions correlating with advanced histological grade,icrovascular invasion, chemoresistance, and shorter sur-

ival [74]. Ets-1, a member of the ETS transcriptor factor,as been shown to be associated with invasion and metas-asis. The average labeling index in HCC is lower than inoncancerous lesions. HCC patients with high ets-1 expres-ion showed better outcomes for disease-free survival thanhose with low ets-1 expression. Lower expression levelsere found in HCCs of high TNM stage, poor differenti-

tion, portal invasion, intra hepatic metastasis, large tumorize, and high Ki-67-LI [75].

.7. Apoptosis

The Bcl-2 family is one of the most characterized groupsf apoptosis mediating factors. Among them, Bax, Bak, and
cl-xS act as promoters of apoptosis and Bcl-2 and Bcl-xLct as apoptosis inhibitors [76]. Bcl-2 expression was notound to be associated with prognosis following resection.owever, Bcl-xL over-expression has been found to indepen-

1 Oncolo

doesFcddLdccp

aecfhfvditas

2

m(armmQplteatftraunfAivsbH

o[

3m

3

ctwnspdii[aIepsiioalsecobefshcnHwwcsdmthEc


ently predict the decreased overall and disease free survivaln multivariate analysis [76,77]. Garcia et al. also foundxpression of Bax to independently predict increased overallurvival following resection of HCC [76]. The expression ofas and Fas ligand (Fas L) also play a role in apoptosis of can-er cells including HCCs and has prognostic significance forisease-free survival [78]. Fas expression level is significantlyecreased in poorly differentiated, large size HCC, while Fasexpression is observed exclusively in moderately or poorly

ifferentiated cases. Overexpression of aberrant programmedell death ligand-I (PD-LI) is recently reported to be asso-iated with higher recurrence and poor prognosis in HCCatients [79].

Survivin belongs to family of anti-apoptotic proteinsnd inhibits apoptosis mainly by targeting the terminalffectors caspase-3 and -7 in the apoptotic protease cas-ade. Its expression is associated with poor prognosisollowing resection of HCC. Nuclear survivin expressionas been shown to be associated with shortened diseaseree survival [80]. Patients with tumors expressing sur-ivin mRNA suffered high rates of recurrence and poorerisease specific survival rates than those tumors not express-ng. High survivin/GADPH mRNA ratio has been showno independently predict the tumor recurrence after hep-tectomy and to be associated with reduced disease freeurvival [81].

.8. Telomerase activity

Human telomerase is a ribonucleoprotein that has threeajor components: human telomerase RNA component

hTERC); human telomerase-associated protein 1 (hTEP1);nd human telomerase reverse transcriptase (hTERT). Recenteports support the concept that activation of telomeraseay be an important and obligate step in the develop-ent of most malignant tumors, including human HCC [82].uantitative analysis of telomerase activity showed that theatients with positive telomerase activity in non-tumorousiver tissue have higher recurrence rate after resection. Highelomerase activity in HCC patients by TRAP assay (telom-ric repeat amplification protocol assay) was found to bessociated with lower 5-year survival rates following resec-ion [83] and to consistently independently predict diseaseree survival on multivariate analysis [84]. The relativeelomerase activity (RTA) of patients with early recur-ence was significantly higher. In a study by Tatsuma etl., telomerase activity was detected in peripheral bloodsing telomerase PCR ELISA assay and was found sig-ificantly higher in HCC patients than the results obtainedor chronic liver disease patients and healthy controls [85].mong the HCC patients, peripheral blood telomerase activ-

ty was significantly higher in the stage III HCC with
ascular invasion than stage II HCC without vascular inva-ion. This suggests that peripheral telomerase activity cane a potential practical diagnostic or predictive marker ofCC for the detection of circulating hepatoma cells in blood
tp[w

gy/Hematology 82 (2012) 116–140

f HCC patients reflecting hematogenous micrometastasis85].

. Cell adhesion and degradation of extracellularatrix

.1. Adhesion molecules and related markers

E-cadherin is a transmembrane glycoprotein that mediatesell–cell adhesion. Beta-catenin protein have multifunc-ional role, either as a regulator of cell–cell adhesionhen complexed to E-cadherin on the membrane or as auclear regulator of gene expression along the Wnt/winglessignal transduction pathway. The Wnt/�catenin signalingathway was involved in homeostasis, cell proliferation,ifferentiation, motility, and apoptosis [86]. This pathways deregulated in number of cancers, including HCC aris-ng from HBV/HCV infections and alcoholic liver cirrhosis87]. The nuclear beta-catenin can be easily assessed onrchival histological tissues including biopsies with a simpleHC technique; however, evaluation of decreased E-cadherinxpression requires comparison with adjacent non-neoplasticarenchyma and, thus, requires larger or multiple biopsyamples [30]. The expression level of E-cadherin correlatesnversely with histological grade and prognosis of HCC andts underexpression might contribute to the early recurrencef HCC [88]; however, Inagawa and colleagues found nossociation between E-cadherin expression and survival fol-owing resection of HCC less than 3 cm in size [89]. A recenttudy demonstrated that serum soluble E-cadherin levels werelevated in patients with HCC and high serum soluble E-adherin (>800 ng/mL) was associated with early recurrencer extrahepatic metastasis [90]. High expression of alpha,eta, and gamma-catenin were correlated with poorly differ-ntiated HCC grade. Significant positive associations wereound between high expression of gamma-catenin and cap-ular invasion or presence of satellite nodules, and betweenigh beta-catenin expression and vascular invasion. However,onflicting results have been reported on prognostic value ofuclear and non-nuclear beta-catenin expression [89,91,92].CCs with nonnuclear type beta-catenin overexpressionere frequently larger than 5 cm in diameter and associatedith poor cellular differentiation, invasiveness, and signifi-

antly shorter disease-free survival [92]. Fiorentino et al. havehown low E-cadherin and nuclear beta-catenin as indepen-ent predictors of recurrence of disease in both univariate andultivariate analysis [30]. A retrospective study analyzing

he Wnt-1 protein expression in 63 HCC patients found thatigh tumor Wnt-1 expression was associated with diminished-cadherin expression and increased tumor recurrence afterurative resection [93]. It may be more exact and valuable
o detect the co-expression of E-cadherin/catenin complex inredicting tumor invasion, metastasis, and patient’s survival94]. A retrospective study of eighty-three patients with HCCho underwent OLT reported that the concurrent detection

Oncolo

obds

rClcosfmtvsohnCrdfswntDmgsfh[

oGapetobacaolttalentv

lissath[hHponauimTcaipefa

3

fdiemi[wytaopplt

twImw[


f high MIB-1 index and low equal E-cadherin and nucleareta-catenin positivity in a HCC patient may be able to pre-ict the recurrence of disease with a probability of 99% andingle may detect by 88% [30].

Osteopontin, a cell-adhesion molecule, plays an importantole in tumor progression and metastasis through binding toD44 and integrin. Its role has been found in inflammatory

iver disease and HCC. Retrospective analysis of 125 surgi-ally resected HCC specimens found that the overexpressionf osteopontin independently correlates with vascular inva-ion and thus predicts poor survival [95]. The CD44 proteinsorm a ubiquitously expressed family of cell surface adhesionolecules involved in maintaining organ and tissue struc-

ure via cell–cell and cell–matrix adhesion; however, certainariant isoforms can also mediate lymphocyte activation, pre-entation of chemical factors, and hormones. The expressionf multiple CD44 isoforms is greatly upregulated in variousuman cancers and may be useful as a diagnostic or prog-ostic marker [96]. Up-regulation of CD 44 isoforms such asD44s, CD44v5, CD44v6, CD44v7–8, and CD44v10 cor-

elates with histological grade, being the highest in poorlyifferentiated HCC. Positivity for one or more CD44 iso-orms was the most useful independent factor for overallurvival [97]. A recent study reported that the HCC patientsith combined positivity for osteopontin and CD44 have sig-ificantly lower overall and disease free survival comparedo than those of OPN-negative, CD44-negative patients [98].etection of serum concentration of intercellular adhesionolecule-1 (sICAM-1) may be a marker for disease pro-

ression and predicts tumor recurrence after surgery. Manyeparate studies have shown that higher sICAM-1 levels wererequently observed in patients with multiple lesions, intra-epatic metastasis, and were associated with poor prognosis22,99].

RhoC-GTPase, a member of the Ras superfamilyf guanosine triphosphatases (GTPases) exhibits intrinsicTPase activity and its activation leads to the assembly of the

ctin–myosin contractile filaments into focal adhesion com-lexes leading to cell polarity facilitating motility. Recently,merging data suggest that RhoC gene may have a poten-ial role in carcinogenesis of tumor cells as overexpressionf RhoC was found in metastatic lesions of inflammatoryreast cancer [100], pancreatic ductal adenocarcinoma [101]nd ovarian carcinoma [102]. Increased expression of RhoCould result in: (a) disruption of cell polarity, which playsn important role in the epithelial–mesenchymal transitionbserved in more aggressive tumors; (b) contribution to theoss of adherens junctions; (c) increase motility and abilityo remold the extra-cellular matrix, which is required forumor cells to become locally invasive; and (d) increase inngiogenic factors that would result in promotion of vascu-arisation in tumor and increase the likelihood of tumor cell
ntering the bloodstream [103]. Wang et al. reported the sig-ificantly higher expression of RhoC in HCC tissues than inhe corresponding pericancerous liver tissue and found ele-ated expression to correlate positively with later events of
s((P

gy/Hematology 82 (2012) 116–140 123

iver carcinogenesis including poor cell differentiation, veinnvasion, number of tumor nodes, and extrahepatic metasta-is. They also suggested that western blotting has a betterensitivity than anti-RhoC immunohistochemical staining,nd RhoC expression can be used as a potential prognos-ic marker for HCC patients as RhoC-negative HCC patientsad a better prognosis than the RhoC-positive HCC patients103]. Deleted in liver cancer 2 (DLC2), a unique RhoGAP,as been recently identified as a tumor suppressor gene inCC. A study on 128 HCC patients reported that the underex-ression of DLC2 in HCC correlates with cell differentiation,verexpression of RhoA, and is associated with poor prog-osis [104]. End binding protein 1 (EB1) has been identifieds a protein bound to the APC tumor suppressor gene prod-ct. EB1 is controlled by c-myc, RhoA, and CDC42. EB1nhibits the ability of APC to bind the actin filaments, which

ay be required for maintenance of cell–cell adhesion [105].he interaction of EB1 and APC may play a key role in theytoskeleton organization, cell migration and proliferation,nd its aberrant regulation could affect the malignant behav-or of HCC, probably resulting in poor prognosis for HCCatients. Immunohistochemistry in 145 cases revealed thatxpression of EB1 significantly correlated with poorly dif-erentiated HCC and a poor prognostic factor for recurrencefter curative surgery [106].

.2. Degradation of extracellular matrix

The matrix degrading metalloproteinase’s (MMPs) are aamily of proteolytic enzymes characterized by their ability toegrade the extracellular matrix (ECM) and play crucial rolen the process of cancer invasion and metastasis. Elevated lev-ls of MMP-2 (gelatinase-A), which degrades collagen IV, aain component of the basement membrane, have been found

n patients who suffered with early recurrence post-resection107]. Concomitant over-expression of MMP-7 with MMP-2as associated with recurrence within the first postoperativeear [108]. The presence of higher content of MMP-9 in HCChan that in surrounding liver parenchyma can also be useds an important index to judge the invasion and metastasisf HCC. Additionally, plasma MMP-9 levels can reflect theotential and ongoing activity of vascular invasion in HCCatients [109]. Expression of human macrophage metalloe-astase (MMP-12) was also found to independently predicthe improved overall survival on multivariate analysis [110].

Urokinase plasminogen activator (uPA) is a serine pro-ease that converts plasminogen into the protease plasmin,hich subsequently degrades the ECM and activates MMP.

ncreased uPA activity (assessed by ELISA) may be theost sensitive factor affecting HCC invasion and associatedith reduced disease free survival and recurrence of HCC

111,112]. Zheng and colleagues showed combined expres-
ion of urokinase plasminogen activator (uPA), its receptoruPAR), and its inhibitor plasminogen activator inhibitor-1PAI-1) to be associated with reduced overall survival. TheAI-1 protein, a multifaceted proteolytic factor functions as

1 Oncolo

artpmwIttLHb

4

scummsfiggeibtr[

4

aCaffrsadcudsd

4

it

phbcmted[wiptasoeias

4(

avNiealIlhr

4

tciaisfb

4


n inhibitor of the protease uPA, and plays an importantole in signal transduction, cell adherence, and cell migra-ion. Although, it inhibits uPA during blood coagulation, butaradoxically, it promotes invasion and metastasis. In manyalignancies including HCC, elevated PAI-1 is associatedith tumor aggressiveness and poor patient outcome [112].

n the last few years, Giannelli and colleagues have consis-ently found a3b1 integrin, the main receptor for Ln-5 in theissue specimens obtained from aggressive and invasive HCC.n-5, a member of the Ln family that expressed in “de novo”CC, absent in the peri tumoral tissue, and was reported toe involved in metastatic spread of HCC [13].

. Angiogenesis associated markers

HCC is typically a hypervascular tumor receiving its bloodupply mainly from hepatic artery. Tumor angiogenesis isritical to both growth and metastasis of cancer, and is reg-lated by angiogenic factors. In recent years, intratumoricrovessel density (MVD) and many angiogenesis-relatedarkers were found to be related with prognosis of HCC

uch as vascular endothelial growth factor (VEGF), basicbroblast growth factor (b-FGF), platelet derived endothelialrowth factor (PD-EGF), thrombospondin (TSP), angio-enin, pleiotrophin and endostatin (ES). They have beenvaluated and could be used to determine the risk of develop-ng cancer, screening for early detection, distinguish betweenenign and malignant disease. In established malignancies,hey can be used to determine prognosis, to predict theesponse to therapy, and to monitor the clinical course109,113].

.1. Microvessel density (MVD)

The intratumor MVD is an index of tumor angiogenesisnd can be visualized by immunohistochemical staining withD34, CD31, von Willebrand factor (vWF), Factor VIII, andlpha smooth muscle actin [114]. MVD using CD34 has beenound to be independently prognostic for decreased diseaseree survival on multivariate analysis in patients undergoingesection of HCC under 5 cm in size [115]; however, anothertudy suggested no prognostic value [116]. Similarly, MVDssessed using vWF has been shown to independently pre-ict decreased disease free survival [117]; however, Poon andolleagues in a large study found no prognostic associationsing the same marker [115]. These differing results might beue to the differing endothelial markers, sampling site, andelection of hotspots to define MVD; however, it remainsebatable whether MVD offers any prognostic value.

.2. Vascular endothelial growth factor (VEGF)

VEGF is a key angiogenic stimulator and many stud-es have demonstrated strong association between elevatedumor expression of VEGF and advanced disease or poor

woNe

gy/Hematology 82 (2012) 116–140

rognosis in various cancers. VEGF overexpression wasigher in HCC patients with early recurrence and haseen associated with metastatic recurrence [28]. Jeng andolleagues analyzed the expression of VEGF165 isoformRNA in resected HCC specimens using reverse transcrip-

ase polymerase chain reaction (RT-PCR) and found positivexpression of VEGF165 mRNA to independently predict theevelopment of recurrence and recurrence related mortality118]. Over-expression of VEGF was correlated significantlyith p53 expression in 38.3% HCC cases, and suggested as an

ndependent prognostic marker for high recurrence rate andoor prognosis [23]. Serum levels of VEGF have been foundo correlate with VEGF expression in tumor and may evenllow the assessment of prognosis preoperatively [119]. Higherum levels of VEGF correlates significantly with absencef tumor capsule, presence of intrahepatic metastasis, pres-nce of microscopic venous invasion, advanced stage, andt may be useful as a biologic marker of tumor invasivenessnd independently predicts decreased disease free and overallurvival on multivariate analysis [119–121].

.3. Platelet-derived endothelial cell growth factorPD-EGF)

PD-EGF, also known as thymidine phosphorylase, is a pro-ngiogenic factor known to play a role in angiogenesis andenous invasion in hepatitis C virus associated HCC [122].o studies have found a correlation between PD-EGF activ-

ty in HCC tissue and survival; however, increased PD-EGFxpression in normal liver adjacent to HCC has been associ-ted with a reduced disease free survival and more frequentong term recurrence (>24 months post hepatectomy) [123].n patients with even alpha-fetoprotein negative hepatocel-ular carcinoma, high co-expression of VEGF and PD-EGFas been reported to predict the poor prognosis after curativeesection [124].

.4. Hypoxia-inducible factor

Hypoxia inducible factor-1 (HIF-1) is a basic helix PASranscription factor that plays a role in angiogenesis. HIF-1aan activate several genes including erythropoietin, PD-EGF,NOS, and VEGF. Overexpression of HIF-1a has been associ-ted with resistance to chemotherapeutics and poor prognosisn some cases. Wada et al. found nuclear HIF-1a expres-ion >1% to be significantly associated with reduced diseaseree survival following resection of HCC; however, this hadorderline significance on multivariate analysis [125].

.5. Nitric oxide synthase (NOS)

NOS is the rate limiting step in synthesis of nitric oxide,
hich plays an important role in tumor angiogenesis. Nitricxide synthase (NOS) exists as three isoforms: endothelialOS, neuronal NOS and inducible NOS (iNOS). Over-
xpression of iNOS has been identified in various human

Oncolo

mnbtteis

4

aebpaCu

4

ibaefo

4

iIlXtfr

4

famticspaf1h

w[rpm8sIosabo

4

wtwAOpsohssfptsgiv

5

5

mtiampacs


alignancies and found to be associated with aggressive phe-otype and poor prognosis. Ikeguchi et al. found associationetween iNOS overexpression in HCC and increased risk ofumor recurrence [126]. Rahman and colleagues suggestedhat iNOS expression alone has no prognostic value; how-ver, combined negative expression of iNOS and COX-2 canndependently predict the improved disease-free and overallurvival following hepatectomy [127].

.6. Basic fibroblast growth factor

Basic fibroblast growth factor (b-FGF) is a soluble hep-rin binding polypeptide with a potent mitogenic effect onndothelial cells. Poon and colleagues found that levels of-FGF above the median of (>10.8 pg/mL) independentlyredicted the decreased disease free survival on multivari-te analysis in a series of 88 HCC patients [128]. However,hao et al. failed to identify any association with prognosissing even a lower cut off (>2.1 pg/mL) [120].

.7. Tissue factor

Tissue factor (TF) is a transmembrane glycoproteinnvolved in triggering the extrinsic coagulation pathway byinding with factor VII. Its expression was associated withngiogenesis and correlate with metastatic potential in sev-ral human malignancies [16]. High TF expression has beenound to independently predict the decreased disease-free andverall survival following resection of HCC [129].

.8. Endostatin/collagen XVIII

Endostatin is a potent angiogenesis inhibitor, specificallynhibiting the proliferation and migration of endothelial cells.n a study of 105 resected HCC specimens, Hu and col-eagues shown a higher expression of endostatin/collagenVIII in adjacent non-tumor tissue and a significant associa-

ion was reported for shorter overall and disease free survivalollowing resection and independently predicting the tumorecurrence [130].

.9. Interleukin-8

IL-8 is a multifunctional interleukin, belongs to the super-amily of CXC chemokines that has shown to be a potentngiogenic both in vivo and in vitro [131]. Several experi-ental studies have demonstrated that IL-8 mRNA increases

he activity of some MMPs and collagenase, thus increas-ng the invasiveness of tumor cells suggesting an importantorrelation with metastatic potential, such as vessel inva-ion [132]. IL-8 is expressed in both the tumor and serum ofatients with HCC. Low preoperative serum IL-8 levels were
ssociated with significantly improved overall and disease-ree survival [133]. Akiba et al. reported IL-8 expression in00% of clinical cases of HCC and found the significantlyigher incidence of portal and venous invasion in patients
lpda

gy/Hematology 82 (2012) 116–140 125

ho expressed more IL-8 at tumor sites than normal liver134]. In a recent study, IL-8 expression was measured usingeverse transcriptase-polymerase chain reaction in 45 HCCatients who underwent surgical resection. The incidence oficroscopic vessel invasion was significantly higher in IL-

-positive than in IL-8-negative tissues irrespective of tumorize. Higher IL-8 expression was noted at pathologic stageII/IV than in those at stage I/II. A simultaneous in vitro effectf IL-8 on HepG2 development by using fluorometric assayshowed that IL-8 stimulates HepG2 chemotactic and invasivectivities rather than cell proliferation [135]. Anti–IL-8 mighte an alternative treatment strategy against the progressionf human HCC through inhibiting vessel invasion.

.10. Angiopoietins (Ang-1 and Ang-2)

Angiopoeitins are endothelial cell growth factors,hich act as ligands for the tyrosine kinase recep-

or. Angiopoietin-1 (Ang-1) promotes vascular maturation,hereas angiopoietin-2 (Ang-2) acts as an antagonist ofng-1 and induces angiogenesis in the presence of VEGF.ver-expression of Ang-2 has been associated with poorrognosis in various human cancers including HCC. Atudy investigated immunohistochemical expression of vari-us angiogenesis associated markers (VEGF, angiopoietins,ypoxia-induced factor-1alpha, thrombospondin-1) in 60pecimens of surgically resected HCC and found that Ang-2taining had a significant correlation with the grade of dif-erentiation of HCC cells and Ang-2 expression correlatedositively with microvessel density. The disease-free survivalime of patients with high Ang-2 and/or HIF-1alpha expres-ion was significantly shorter than that of the low expressionroup [125]. The ratio of Ang-2:Ang-1 was found to be anndependent prognostic factor for overall survival on multi-ariate analysis [136].

. Growth factors and their receptors

.1. Transforming growth factor-beta (TGF-βs)

TGF-�s belong to a superfamily of polypeptide signalingolecules involved in regulating cell growth, differentia-

ion, angiogenesis, invasion, and immune function. TGF-�1s a predominant form in humans promoting angiogenesisnd correlates with cellular immunosuppression. TGF-�1RNA and its protein were over-expressed in HCC com-

ared with surrounding liver tissues, especially in small-sizednd well-differentiated HCCs [137]. However, Ikeguchi andolleagues found no relationship between TGF-� expres-ion and post-hepatectomy survival [138]. Serum TGF-�1
evel has been shown to be elevated in HCC patients com-ared to healthy adults or patients with non-malignant liverisease [139,140]. Elevated urinary levels of TGF-�1 werelso suggested as a prognostic marker, although levels were

1 Oncolo

mw

5

(wtodraTncvo

5

ck3obas

5

arpeblstst

6

6

pclher

Tuattuiibo(tiont(po

sptdvoeciAcfnLAieaifirLmHa

6

fgw


easured only at the time of diagnosis and no patients under-ent surgery [141].

.2. Tumor-specific growth factor (TSGF)

Malignant tumor releases tumor-specific growth factorTSGF) into peripheral blood during its growing period,hich results in blood capillary amplification surrounding

he tumor. Thus, serum levels of TSGF reflect the existencef tumor. It has been indicated that TSGF can be used as aiagnostic marker in detecting HCC, and its sensitivity caneach 82% at the cut-off value of 62 U/mL and may have

higher accuracy with the simultaneous determination ofSGF and other tumor markers. The simultaneous determi-ation of TSGF (at the cut-off value of 65 U/mL), AFP (at theut-off value of 25 ng/mL), and serum ferritin (at the cut-offalue of 240 �g/mL) has a sensitivity of 98.4% and specificityf 99% [140].

.3. Epidermal growth factor receptor family

The epidermal growth factor receptor (EGFR) familyonsists of four closely related transmembrane tyrosineinase receptors: EGFR (erbB-1), c-erb-2 (Her-2/neu), c-erb-(HER-3), and c-erb-4 (HER-4). These bind with ligands

f the EGF family, including EGF, TGF-alpha, and heparininding EGF. High levels of EGFR expression have beenssociated with early recurrence and reduced disease freeurvival following resection [109,142].

.4. Hepatocyte growth factor/scatter factor

Hepatocyte growth factor/scatter factor (HGF/SF) isn important humoral mediator of liver regeneration andelated to molecular mechanisms of hepatocarcinogenesis viaaracrine system involving its cellular receptor, c-met. C-metxpression was higher in invasive type of HCC as determinedy gross type, vessel invasion, intrahepatic metastasis, histo-ogical type, and has been associated with reduced overallurvival [22,109,143] and might be important to caution forhe presence of intrahepatic metastasis. A significantly highererum HGF levels were found in patients with invasive grossype or intrahepatic metastasis positive tumors [143].

. Oncofetal and glycoprotein antigens

.1. Alpha-fetoprotein and alpha-fetoprotein-L3

Alpha fetoprotein (AFP) is a fetal specific glycoproteinroduced primarily by the fetal liver. Normally, its serum con-entration falls rapidly after birth and its synthesis in adult
ife is repressed. However, more than 70% of HCC patientsave a high serum concentration of AFP because of the tumorxcretion. Even forty years after its discovery, serum AFPemains a most useful tumor marker for screening of HCC.
paai

gy/Hematology 82 (2012) 116–140

he serum concentration of 20 ng/mL is the most commonlysed cut-off value to differentiate HCC patients from healthydults and a level > 400 ng/mL is usually regarded as diagnos-ic. However, two thirds of HCC patients with the nodule lesshan 4 cm have serum AFP levels less than 200 ng/mL andp to 20% of HCC patients do not produce AFP [140]. Somenvestigations have showed that the cut-off value is fluctuantn different ethnic groups. The best cut-off value of AFP haseen reported to be 30 ng/mL (sensitivity of 65%, specificityf 89%) in Sicilian population compared with 200 ng/mLsensitivity of 70%, specificity of 100%) in Burman popula-ion [140,144]. One of the possible reasons for this differences the diverse living circumstance, which has a great influencen epidemiology. The positive predictive value of AFP is sig-ificantly lower in detecting HCC patients with viral etiologyhan that in detecting HCC patients with non-viral etiology70% vs. 94%, p < 0.05), and it will not reach 100% in HCCatients with viral etiology unless their serum concentrationf AFP is greater than 400 ng/mL [140,144].

Besides the purpose of screening HCC, serum and tis-ues AFP could also be used as prognostic indicators. HCCatients with a high AFP concentration (≥400 ng/mL) tendo have greater tumor size, bilobar involvement, massive oriffuse types, portal vein thrombosis, and a lower median sur-ival rate [145,146]. This is partially caused by the expressionf ephrin-A1 (an angiogenic factor) and the ability of AFP tolicit the escape of carcinoma cells from the host’s lympho-ytes immune surveillance [147]. Total AFP can be dividednto three different glycoforms, namely AFP-L1, AFP-L2 andFP-L3, according to their binding capability to lectin lens

ulinaris agglutinin (LCA). AFP-L1, as the non-LCA-boundraction, is the major glycoform of AFP in the serum ofonmalignant hepatopathy patients. On the contrary, AFP-3, as the LCA-bound fraction, is the major glycoform ofFP in the serum of HCC patients, and it can be detected

n approximately 35% of patients with small HCC (<3 cm),specially when the tumor mass is supplied by the hepaticrtery. At the cut-off level of 15%, sensitivities of AFP-L3n detecting HCC range from 75% to 96.9% with speci-cities of 90–92.0% correspondingly [140]. Some clinicalesearches have indicated that the high percentage of AFP-3 is closely related to poor differentiation and biologicallyalignant characteristics (especially portal vein invasion) ofCC [148], and worse liver function, poorer tumor histology,

nd larger tumor mass [149].

.2. Glypican-3

Glypican-3 (GPC3), a membrane-anchored heparan sul-ate proteoglycan, has been demonstrated to interact withrowth factors and modulate their activities. GPC3 mRNAas upregulated significantly in tumor tissues of HCC com-
ared to paraneoplastic liver tissue, liver tissues of healthydults, and liver tissues of patients with nonmalignant hep-topathy. Nassar et al. suggested that GPC-3 can be usedn fine needle aspiration biopsies (FNABs) in differentiat-

Oncolo

ilssinHAItosacGwn[

7

7

alT�gooHso

7

fcHp(ccIA7Adtab8a

wm[

7

auoccipasiia(drcstftEsscogopcacDqtasiTtpfeorr


ng primary malignant tumor from benign and preneoplasticiver lesions, and metastatic carcinoma [150]. The expres-ion of GPC3 (at both mRNA and protein levels) in theerum of HCC patients was significantly higher than thatn the serum of healthy adults or patients with nonmalig-ant hepatopathy [151]. It can be detected in 40–53% ofCC patients and 33% of HCC patients seronegative for bothFP and Des-gamma-carboxyprothrombin (DCP) [151,152].

t has been shown that soluble GPC3 (sGPC3), the NH2-erminal portion of GPC3, is superior to AFP in the sensitivityf detecting well or moderately differentiated HCC, and theimultaneous determination of both markers improves over-ll sensitivity from 50% to 72% [151,153]. Recently, a studyompared the survival rate between the GPC3-positive andPC3-negative HCC patients. GPC3 positivity correlatedith poor prognosis and identified as an independent prog-ostic factor for the overall survival on multivariate analysis154].

. Enzymes and isoenzymes

.1. Gamma-glutamyl transferase

Serum gamma-glutamyl transferase (GGT) in healthydults is mainly secreted by hepatic Kupffer cell and endothe-ial cell of bile duct, and its activity increases in HCC tissues.otal GGT can be divided into 13 isoenzymes (I, I′, II, II′,, �, �, �A, VIIB, �C, �A, �B) by using polymeracrylamideradient gel electrophoresis, and some of them (I′, I, II′) cannly be detected in the serum of HCC patients. Sensitivitiesf GGTII have been reported to be 74.0% in detecting largeCC and 43.8% in detecting small HCC. Sensitivity can be

ignificantly improved with the simultaneous determinationf GGTII, DCP, and AFP [155].

.2. Alpha-l-fucosidase

Alpha-l-fucosidase (AFU) is an enzyme to hydrolyzeucose glycosidic linkages of glycoprotein and gly-olipids. Its activity increases in the serum ofCC patients (1418.62 ± 575.76 nmol/mL/h) com-ared with that in the serum of healthy adults504.18 ± 121.88 nmol/mL/h, p < 0.05), patients withirrhosis (831.25 ± 261.13 nmol/mL/h), and patients withhronic hepatitis (717.71 ± 205.86 nmol/mL/h [156,157].t has been reported that the sensitivity and specificity ofFU at the cut-off value of 870 nmol/mL/h were 81.7% and0.7%, respectively, in contrast with 39.1% and 99.3% ofFP at the cut-off value of 400 ng/mL, and the simultaneousetermination of both markers can improve the sensitivityo 82.6% [156]. This indicates that AFU could serve as

valuable supplementary to AFP in the detection. It haseen indicated that HCC will develop within few years in2% of patients with liver cirrhosis, if their serum AFUctivity exceeds 700 nmol/mL/h. The activity of AFU

qDtp

gy/Hematology 82 (2012) 116–140 127

as reported to be elevated in 85% of patients at least 6onths before the detection of HCC by ultrasonography

157].

.3. Des-gamma-carboxyprothrombin (DCP)

DCP, also known as a protein induced by vitamin Kbsence or antagonist II (PIVKA-II), is an abnormal prod-ct from liver carboxylation disturbance during the formationf thrombogen, and acts as an autologous mitogen for HCCelllines [158]. Its mean serum concentration, which is notorrelated to serum levels of AFP, is obviously elevatedn HCC patients compared with that in healthy adults andatients with non-malignant hepatopathy [159,160]. Thoughfew researches have an opposite result, serum and tis-

ues DCP have been proved to be more useful than AFPn differentiating HCC from nonmalignant hepatopathy andn detecting patients with small HCC [146,159,160]. Cui etl. reported that the sensitivity and specificity of serum DCPat the most commonly used cut-off value of 40 mAU/mL) iniscriminating HCC from cirrhosis were 51.7% and 86.7%,espectively, which were much better than those of AFP at theut-off value of 20 ng/mL [159]. Another study reported theensitivity and specificity of 89% and 86.7% respectively (athe cut-off value of 125 mAU/mL) in discriminating HCCrom nonmalignant hepatopathy, which were much betterhan those of AFP at the cut-off value of 11 ng/mL [160].lectrochemiluminescence enables even measurement of lowerum concentration of DCP (high-sensitive DCP), and theimultaneous determination of high-sensitive DCP (at theut-off value of 40 mAU/mL), AFP (at the cut off valuef 20 ng/mL), and AFP-L3 (at the cut-off value of 10%)ive the highest accuracy (sensitivity of 82.1%, specificityf 82.4%, and accuracy of 82.2%) [161]. Besides the pur-ose of screening HCC, serum DCP could also be used as alinicopathological or prognostic indicator for HCC patients,nd may be more useful than AFP in reflecting the invasiveharacteristics of HCC. It has been reported that patients withCP seropositive and AFP seronegative have a higher fre-uency of HCC with a distinct margin, large nodule morehan 3 cm, few nodules, moderately to poorly differentiation,nd portal vein infiltration [162,163]. A recent prospectivetudy reported serum PIVKA-II level, not serum AFP, as anndependent prognostic factor in HBV related HCC [164].he simultaneous determination of serum DCP levels and

issue DCP expression was more useful in predicting therognosis and early recurrence of HCC patients than eitheractor alone, especially in patients with low serum DCP lev-ls [165,166]. High serum DCP levels can also be a predictorf prognosis following a particular treatment modality. Aetrospective analysis of 199 HCC patients who underwentesection and 209 HCC patients who underwent radiofre-
uency ablation (RFA) suggested that among patients withCP > 100 AU/L, treatment procedure was a significant fac-
or for survival and recommended surgical resection for suchatients [167].

1 Oncolo

7

aifsGsmpo

8

8

oMbp[orbcpHiH1v2twtd[

8

mtootcaaTpds

mSGarisraGcodpp

8

sdpmt6nmo5t

8

rcsaliptasr7

8

hw


.4. Golgi phosphoprotein 2

Golgi phosphoprotein 2 (GOLPH2), a Golgi apparatusssociated protein, has been shown to have a higher sensitiv-ty than AFP in the detection of HCC [168]. A recent studyound that GOLPH2 protein was highly expressed in tis-ues of HCC (71%) and bile duct carcinoma (85%) patients.OLPH2 protein levels were detectable and quantifiable in

era by ELISA. In patients with hepatitis-C, serial ELISAeasurements in the course of the disease appear to be a

romising complimentary serum marker in the surveillancef HCC [169].

. Genetic biomarkers

.1. Alpha-fetoprotein mRNA (AFP mRNA)

The presence of circulating HCC cells may be indicativef metastasis if AFP mRNA is detected in peripheral blood.atsumura et al. first reported that single HCC cell could

e detected in circulation by means of reverse-transcriptionolymerase chain reaction (RT-PCR), targeting AFP mRNA170]. Subsequently, many investigators reported the valuef AFP mRNA as a predictor for HCC recurrence, but theesults were rather controversial. This may be due to thelood-borne dispersion of both tumor cells and normal liverells and the mis-transcription of mRNA encoding AFP byeripheral mononuclear cells. The recurrence-free interval ofCC patients with postoperative serum AFP mRNA positiv-

ty has been reported to be significantly shorter than that ofCC patients with postoperative negativity (53% vs. 88% atyear, 37% vs. 60% at 2 years, p = 0.014) [171] and (52.6%s. 81.8% at 1 year, 15.6% vs. 54.5% at 2 years, and 0% vs.9.2% at 3 years, p < 0.001) [172]. A meta-analysis showedhat the expression of AFP mRNA one week after surgeryas correlated with the recurrence of HCC [173]. Its sensi-

ivity and specificity can be increased with the simultaneousetermination of melanoma antigen gene (MAGE-1) mRNA140].

.2. Gamma-glutamyl transferase mRNA (GGT mRNA)

Gamma-glutamyl transferase (GGT) is a plasmaembrane-bound enzyme, widely distributed in mammalian

issues catalyzing hydrolysis of glutathione and transferf �-glutamyl residue. At least four different GGT genesr pseudogenes are present in human genome; however,he expression is usually monogenic [174]. GGT mRNAan be detected in the serum and liver tissues of healthydults, patients with liver disease, benign liver tumor, HCC,nd secondary tumors of the liver [140]. According to
sutsumi’s report, type A GGT mRNA was commonlyresent in normal liver and remains same in benign liveriseases. Type B was the main type in cancerous tissueuggesting that changes in the expression of hepatic GGT
ftin

gy/Hematology 82 (2012) 116–140

RNA may be related to the development of HCC [175].heen et al. demonstrated that patients of HCC with type BGT mRNA both in cancer and in non-cancerous tissue hadworse outcome, earlier recurrence, and more recurrence

elated mortality. The presence of type B GGT mRNAn cancerous tissue was statistically correlated with higherum level of AFP, daughter nodules, higher post-resectionecurrence rate than those without it (63.6% vs. 14.3%),nd lower post-recurrence survival. The presence of type BGT mRNA in non-cancerous liver tissue was significantly

orrelated with hepatitis C infection, high serum levelf AFP, Edmondson-Steiner grade III and IV of cellularifferentiation, absence of infiltration of capsule, vascularermeation, daughter nodules, post-resection recurrence andost recurrence survival [174].

.3. Insulin-like growth factor II-mRNA (IGF-II)

Recently, abnormal expression of IGF-II mRNA has beenuggested as a useful tumor marker for diagnosis of HCC,ifferentiation, extra-hepatic metastasis, and monitoring ofostoperative recurrence. It has been reported that the deter-ination of serum insulin-like growth factor-II (IGF-II) (at

he cut-off value of 4.1 mg/g, prealbumin) has a sensitivity of3%, specificity of 90%, and accuracy of 70% in the diag-osis of small HCC [176]. It can be a complementary tumorarker to AFP for diagnosis of small HCC. The simultane-

us determination of IGF-II and AFP (at the cut-off value of0 ng/mL) can improve the sensitivity to 80% and accuracyo 88% [177].

.4. Albumin mRNA

Extracellular-based assays (circulating DNA/RNA) wereeported to be better than cell-based assays (circulating tumorells) in detection of preneoplastic lesions and micrometasta-is as plasma levels of circulating cancer-derived nucleic acidre higher than the levels of circulating cancer cells and areess prone to sampling errors. Cheung and colleagues stud-ed the preoperative plasma samples obtained from 72 HCCatients who had undergone liver transplantation and foundhat patients with plasma albumin mRNA level (>14.6) hadsignificantly higher recurrence rate on multivariate analy-

is. High plasma albumin mRNA level predicted the 2-yearecurrence rate with sensitivity and specificity of 73% and0%, respectively [178].

.5. Human telomerase reverse transcriptase mRNA

Human telomerase reverse transcriptase (hTERT) mRNAas been reported to be detectable in the serum of patientsith breast cancer and demonstrated to be a novel marker

or HCC diagnosis. The expression of hTERT mRNA inhe serum of HCC patients is significantly higher than thatn the serum of healthy adults or patients with nonmalig-ant hepatopathy, and the use of newly developed real-time

Oncolo

qmriemaatra[

bpr[cisswgHh

8

no(shUbpdtettf[dYaBspTpls9a

cmcpwothTd

hi(HanmstashipcCgoocecsmtap[maomttatcpmtc


uantitative reverse transcription polymerase chain reactionay improve the effectiveness of determination. It has been

eported that the sensitivity and specificity of hTERT mRNAn detecting HCC are 88.2% and 70.0%, respectively, whichxcel those of conventional tumor markers, such as AFPRNA, AFP and DCP. Expression of serum hTERT mRNA,

ssociated with the serum concentration of AFP, tumor size,nd tumor differentiation degree, may be a valuable indica-or of poor prognosis for HCC patients [179]. In contrast, aecent study found no relationship between overall survivalnd AFP or hTERT mRNA expression in peripheral blood180].

There are some other markers in this category, which coulde used as diagnostic or prognostic indicators for HCC. HCCatients with positive MAGE-1 or MAGE-3 mRNA wereeported to die earlier because of metastasis or recurrence181]. The sensitivity and specificity of serum human cervicalancer oncogene (HCCR) (at the cut-off value of 15 �g/mL)n detecting HCC are 78.2% and 95.7%, respectively. Its sen-itivities could achieve 76.9% in detecting HCC patients witheronegative for AFP and 69.2% in detecting HCC patientsith tumor size less than 2 cm [182]. The over-expression ofranulin–epithelin precursor (GEP) in cancerous tissues ofCC is associated with venous infiltration and early intra-epatic recurrence (p < 0.05) [183].

.6. MicroRNAs

MicroRNAs (miRNAs) are small (22–25 nucleotide long)oncoding RNAs that can effectively reduce the translationf target mRNAs by binding to their 3′ untranslated regionUTR). This occurs through the assembly of an RNA-inducedilencing complex composed of a variety of proteins. If theomology between the miRNA sequence and the target 3′-TR is incomplete, then this complex reduces expressiony blocking translation. If, however, the homology is com-lete, then degradation of the target mRNA can result. Toate, more than 300 distinct human miRNAs capable ofargeting thousand of genes have been identified. miRNAxpression patterns (or signatures) are now known to charac-erize the developmental origins of tumors more effectivelyhan mRNA expression signatures and may provide a use-ul tool for the diagnosis and prognosis of human cancer184]. Using global miRNA expression profile in mouse liverevelopment analyzed by an LNA-based miRNA microarray,amamoto et al, report that a novel miR-500 (miRNA) aspotential candidate for oncofetal HCC biomarker [185].ased on microarray data miR-29 has been shown to be

ignificantly down regulated in HCC tissues, implicating aotential prognostic role of miR-29 in HCC therapy [186].hree miRNAs were found to express higher in the HCC sam-les than in non-tumorous tissues, while five miRNAs had
ower expression in HCC. The prediction accuracy of clas-ifying HCC and non-tumor with these 8 miRNAs reached7.8% [187]. Budhu et al. using a supervised algorithm builtunique 20-miRNA metastasis signature that could signifi-
tfre

gy/Hematology 82 (2012) 116–140 129

antly predict (p < 0.001) primary HCC tissues with venousetastases from metastasis-free solitary tumors with 10-fold

ross-validation. These 20 miRNAs may provide a simplerofiling method to assist in identifying patients with HCCho are likely to develop metastases/recurrence [188]. A setf 19 miRNAs involved in cell division, mitosis, and G(1)–Sransition significantly correlated with HCC disease outcome,igher miRNA expression correlates good survival (p < 0.05).hus, suggesting that miRNA expression may prognosticateisease outcome in HCC [189].

Extensive research on miRNA using HCC cell lineselped in understanding HCC tumorigenesis, thus form-ng useful biomarker for diagnosis or prognosis of HCCFig. 1). Aberrant expression of miR-21 can contribute toCC growth and spread by modulating PTEN expression

nd PTEN-dependent pathways involved in mediating phe-otypic characteristics of cancer cells such as cell growth,igration, and invasion [190]. Significantly high expres-

ion of miR-224 in HCC patients (p < 0.05) on one armhrough unknown process promotes proliferation and otherrm inhibits apoptosis inhibitor-5 (API-5) transcript expres-ion. The dual role of miR-224 may potentiate favoringeritable genetic mutations in tumors, thus reaffirming themportant role of miRNAs in the dysregulation of cellularrocesses during tumorigenesis [191]. A significant inverseorrelation between miR-221 and both CDKN1B/p27 andDKN1 C/p57, cell cycle proteins suggested miR-221 onco-enic function in hepatocarcinogenesis [192]. Further studiesn miR-221 have shown that it is involved in the modulationf Bmf, a proapoptotic BH3-only protein, thus regulating theell proliferation and apoptosis proteins towards tumorigen-sis [193]. Array-based miRNA profiling performed on HCCells showed highly deregulated miR-223 expression and atrong inverse relationship with its downstream target Stath-in 1 (p = .006) in HCC cells [194]. Li et al. have indicated

hat they agreed with 5 of the miRs from Murakami et al.,nd found that miR-125b that suppress the cell growth andhosporylation of Akt to be a prognostic marker of HCC195]. miR-122 found up to 70% of total miRNA in the liver,odulates cyclin G1, thus influences p53 protein stability

nd transcriptional activity and reduces invasion capabilityf HCC-derived cell lines [196]. Bcl-w is a direct target ofiR-122 that functions as an endogenous apoptosis regula-

or in these HCC-derived cell lines [197]. miR-122 is underhe transcriptional control of HNF1A, HNF3A and HNF3Bnd loss of miR-122 results in an increase of cell migra-ion and invasion. From a clinical point of view, miR-122an be used as a diagnostic and prognostic marker for HCCrogression [198]. In 76% of HCC patient’s tissue samplesiR-34a was highly expressed compared to adjacent liver

issue samples. In HCC cell line, miR-34a directly targeted-Met and reduced both mRNA and protein levels of c-Met,
hus blocking cell migration [199]. Females are protectedrom HCC occurrence through higher expression of estrogeneceptor alpha, which is compromised due to miR-18a highxpression in HCC tumors [200]. miR-101 has a downstream

130 A. Singhal et al. / Critical Reviews in Oncology/Hematology 82 (2012) 116–140

F nvasioni anscrip

taebmrBadblasmEitmch2i[ric[lm

wtrw

9

upcstdld

tbfapHp

ig. 1. MicroRNAs as biomarkers involved in survival, proliferation, and in various signaling pathways including apoptosis, cell-cycle check point, tr

arget of v-fos oncogene and it is involved in cell invasionnd migration in overexpressed HCC cell lines [201]. Xut al. show that miR-195 may block the G(1)/S transitiony repressing Rb-E2F signaling through targeting multipleolecules, including cyclin D1, CDK6, and E2F3 [202]. Up-

egulation of miR-143 expression transcribed by NF-kappain HBV-HCC promotes cancer cell invasion/migration

nd tumor metastasis by repression of fibronectin type IIIomain containing 3B (FNDC3B) expression [203]. Bead-ased microarray profiling of HCC tissue, revealed that theevel of miR-338 expression can be associated with clinicalggressiveness, such as, tumor size, Tumor-Node-Metastasistage, vascular invasion and intrahepatic metastasis, thus thisethod could be used in large-scale diagnostic trails [204].vidences indicate that miR-23b can recognize target sites

n the 3′-UTR of uPA and of c-met mRNAs and transla-ionally repress the expression of uPA and c-met decreasing

igration and proliferation abilities in HCC cells [205]. HCCell lines exhibit reduced expression of miR-26a, normallyigh in diverse tissues. Systemic administration of this miR-6a resulted in inhibition of cancer cell proliferation andnduction of tumor-specific apoptosis in HCC mouse model206]. Gao et al. have shown that functional polymorphisms3783553 disrupts a binding site for miR-122 and miR-378,ncreasing IL-1alpha expression in vitro and in vivo, thusontributing to HCC susceptibility in the Chinese patients
207]. From 25 patients, pairs of HCC tissue and adjacentiver tissue, seven miRNAs associated with HCC recurrence.
iR-15b having Bcl-w as the target, which ranked highest

hata

of HCC. MicroRNA regulates the expression of various proteins involvedtion factors, phosphatases, oncogenes, and many more.

as negatively correlated with HCC recurrence, a poten-ial prognostic marker [208]. This newly emerging area ofesearch should unravel novel biomarkers of diagnostic asell as prognostic value in HCC.

. Discussion

Hepatocarcinogenesis is a complex multi-state processsually occurring after many years of chronic hepatitisroviding the mitogenic and mutagenic environments to pre-ipitate random genetic alterations (Fig. 2). Recent evidencesuggest that intrinsic biologic characteristics of the tumor inerms of proliferation and invasiveness are probably due toifferent composition and activity of the microenvironment,eading to very different clinical outcome. Therefore, earlyiagnosis and monitoring of HCC is of utmost importance.

Hepatoma tissues synthesize various tumor-related pro-eins. Among these circulating diagnostic and prognosticiomarkers, serum AFP is the most widely used tumor markeror detection and monitoring of HCC. However, the false neg-tive rate with AFP level alone may be as high as 40% foratients with early stage HCC. Even in patients with advancedCC, the AFP levels may remain normal in 15–30% of theatients. Recent researches have confirmed that circulating
epatoma-specific AFP subfraction L3 and DCP excel AFPlone in differentiating HCC from nonmalignant hepatopa-hy and detecting small HCC. Some tumor markers, suchs hTERT mRNA, have also been indicated to have higher


hepato

avmtIaHwd

ag

ouondmatp

Fig. 2. Molecular markers involved in

ccuracies than AFP. IGF-II has been reported to be morealuable than AFP in the diagnosis of small HCC; however,ore studies are needed to demonstrate its superiority. Fur-

hermore, some other tumor markers, such as GPC3, GGTI, AFU, TGF-�1, and TSGF, that have been indicated to bevailable supplementary to AFP and DCP in the detection ofCC, and some of them even can be detected in HCC patientsith seronegative for both AFP and DCP, the simultaneousetermination of these markers may improve the accuracy.

The ability to predict patients at higher risk of recurrencend with a poor prognosis would help to assign risk anduide surgical and other treatments. Recurrent lesion may

mta

carcinogenesis and tumor metastasis.

riginate in following ways: the continuous growth of resid-al tumor due to incomplete excision; metachronous lesionr unrecognized synchronous multifocal primary tumor;ew primary tumor; or intrahepatic/extrahepatic metastasisue to manipulation during surgery. The identification ofetastatic dissemination or multifocal tumor seems to beunrecognized problem by clinicians. Patients with mul-

ifocal tumor are likely to have a better prognosis than aatient with a metastatic cancer [209]. Currently available
orphologic criteria are unable to differentiate between the
wo. Molecular-genetic approaches identifying karyotypiclterations seem to be the most appropriate to investigate

1 Oncolo

tnmsrtr[bscTma

gwuHdbstMmatpisMacpmusrsmbptemiaucatdaatm

nceodmrrsacttcmotommitoomltdalbsbVim

peorpoamcpiwmi


he clonality of the different nodules: monoclonal multipleodules stem from a common malignant precursor, as inetastatic lesions, whereas different clonalities are an expres-

ion of distinct tumors, as in multifocal HCC [210]. Similaresults have been obtained by different investigators, showinghat multiple HCC nodules are an expression of metastasisather than of multifocal cancer in more than 60% of cases211]. However, in both studies the techniques used could note immediately introduced into clinical care, as it is not rea-onable to perform multiple biopsies in patients to assess theorrect diagnosis based on chromosomal or DNA alterations.hese types of studies are very sophisticated and would needore clinical validation in view of the possibility that some

lterations might not be the rule for HCC of multifocal origin.Research into the molecular biology of hepatocarcino-

enesis has identified a multitude of molecular biomarkersith potential prognostic significance (Table 2). Molec-lar biomarkers could provide additional information forCC metastasis and recurrence to that gained from tra-itional histopathological features. A large number ofiomarkers have been shown to have potential predictiveignificance. One important aspect of this is to detecthe transcripts of tumor-associated antigens (such as AFP,

AGEs, and CK19), which are proposed as predictivearkers of HCC cells disseminated into the circulation

nd for metastatic recurrence. Another important aspect iso analyze the molecular markers for cellular malignancyhenotype, including DNA ploidy, cellular proliferationndex, cell cycle regulators, oncogenes, and tumor suppres-ors (especially p53 gene), as well as telomerase activity.

olecular factors involved in the process of HCC invasionnd metastasis, including adhesion molecules (E-cadherin,atenins, ICAM-1, laminin-5, CD44 variants, osteopontin),roteinases responsible for the degradation of extracellularatrix (MMPs, uPA system), as well as angiogenesis reg-

lators (such as VEGF, intratumor MVD), have also beenhown to be potential predictors for HCC metastatic recur-ence and clinical outcomes. The circulating genetic markersuch as AFPmRNA, TGF-�1-mRNA, GGT mRNA, IGF-IIRNA, and IL 8 from peripheral blood of HCC patients have

een most extensively used in monitoring distal metastasis orostoperative recurrence of HCC. One important new trend iso widely delineate biomarkers with genomic and proteomicxpression with reference to predicting metastatic recurrence,olecular diagnosis, and classification, which has been draw-

ng more attention recently. Body fluid (particularly bloodnd urine) testing for biomarkers is easily accessible and moreseful in clinical patients. The prognostic significance of cir-ulating DNA in plasma or serum and its genetic alterations isnother important direction. More attention should be paid tohese areas in the future. As understanding of tumor biologyeepens, more and more new biomarkers with high sensitivity
nd specificity for HCC metastatic recurrence could be foundnd routinely used in clinical assays. However, the combina-ion of the pathological features and some of the biomarkers
entioned above seems to be more practical up to now.

alc[

gy/Hematology 82 (2012) 116–140

Studies have shown inconsistencies and variation in sig-ificance of biomarkers as majority of the studies wereorrelative, retrospective, and focused on individual mark-rs with differing methodologies. These studies include HCCf different etiologies; variation in their prevalence betweenifferent populations may cause differing effects on theolecular pathways involved, may also account for variable

esults. Therefore, a particular marker prognostic for HBV-elated HCC, may be not relevant for HCV-related HCC;uggesting a need of appropriate subanalysis. However, suchnalysis is often not possible for variety of reasons such asombination of etiological factors. Furthermore, malignantransformation may occur regardless of the etiologic agenthrough a pathway of increased liver cell turnover induced byhronic liver injury and regeneration in a context of inflam-ation, immune response, and oxidative DNA damage [3]. In

rder to formulate panels of biomarkers as an important tool,here is a need for both standardized approach for assessmentf molecular biomarkers, and validation of these potentialarkers in larger cohorts of patients. Combing panels ofolecular biomarkers with more traditional histopatholog-

cal characteristics may further enhance their prediction inhose at high risk of disease progression and recurrence. Mostf these markers were studied on post resection specimenr post transplant liver explants; however, identifying thesearkers or features on preoperative biopsy has significant

imitations. First, all tumors are not recognized preopera-ively, secondly, it is not practical or feasible to biopsy alletected tumors multiple times in the preoperative setting,nd thirdly, needle biopsy is a poor predictor for some featuresike micro or macro vascular invasion. Recently, emphasis haseen placed on the role of circulating serum biomarkers asome of the biomarkers could be detected both in tissue andody fluids like serum, urine, and bile (preoperative serumEGF) which are easily accessible and useful in “predict-

ng” the possibility of “early diagnosis” of recurrence andetastasis (Table 3).These markers also allow in formulating preoperative

rognostic criteria to identify patients most likely to ben-fit from particular therapies, such as hepatic resectionr transplantation, as well as predict those most likely toespond to chemotherapeutic agents. The ability to stratifyatients’ prognosis pre-operatively would improve provisionf patient’s information, allow assessment of definitive ther-py or need for any adjuvant therapies, reduces morbidity,ortality, and cost. Realisation of these aims in a clini-

al setting has been made more viable by microarray generofiling; however, much data have been available only assolated studies focusing on specific molecular target or path-ay. This microarray-based technology allows the study ofultiple genes simultaneously, and provides important find-

ngs relating to carcinogenic process. In addition, microarray
nalysis of tissue may enable to predict which patients areikely to respond to specific chemotherapeutic agents, orombinations, thus creating individualized treatment regimes212].


Table 2Prognostic potential of molecular markers in HCC.

Type of marker Poor prognosis Good prognosis

Proliferating activity PCNA-LI, Ki-67-LI, MIB-1, CAS –Nuclear morphology Larger nuclear area –p53 and related genes p53 mutation, expression of MDM2 and p73 –Genomic instability CSI, LOH, MSI-H, DNA aneuploidy –Cell cycle regulators Overexpression of cyclin A, D1, E and methylation of p15, p16 Expression of p27, p57, p18Tumor promotor genes c-myc amplification ets-1 overexpressionApoptosis Overexpression of Bcl-xL, Fas L, survivin Expression of Bax, FasTelomerase activity High LowAdhesion molecules Underexpression of E-cadherin, overexpression of alpha, beta, gamma

catenin, overexpression of osteopontin, upregulation of CD-44 isoforms,RhoC expression

E-cadherin overexpression

ECM degradation Overexpression of MMP-2, MMP-7, MMP-9, uPA, PAI-1, Ln-5 –Angiogenesis related Increase MVD, overexpression of VEGF, PD-EGF, HIF-1a, iNOS, TF,

IL-8, Ang-2–

Growth factors Overexpression of TGF-�, EGFR, c-met –Genetic biomarkers Expression of Type B GGT mRNA, IGF-II mRNA, –MicroRNAs miR18a, miR21, miR101, miR221,miR224, miR143, miR338 miR15b, miR23b, miR26a, miR29, miR34a,

miR122, miR125b, miR195, miR378

PCNA-LI: proliferating cell nuclear antigen labeling index, MIB-1: mindbomb homolog 1, CAS: Chromosome Segregation gene homolog, nm-23: non-metastatic protein-23, CSI: chromosomal instability LOH: loss of heterozygosity, MSI: microsatellite instability, ECM: extracellular matrix, MMP: matrixmetalloproteases, uPA: urokinase plasminogen activator, MVD: microvessel density, VEGF: vascular endothelial growth factor, PD-EGF: platelet-derivede nitric oA rmal grI

ai[rikaeItm

itrshia[f

TP

T

pGTAEAGGTEIM

Dltmg

ndothelial growth factor, HIF-1�: hypoxia-inducible factor-1 alpha, NOS:ng: angiopoeitin, TGF-�: transforming growth factor beta, EGFR: epide

GF-II: insulin-like growth factor-II, miR: microRNA.

Targeted therapy which specifically inhibits molecularbnormalities has emerged as a novel approach for thennovative and effective medical treatment of malignancies213–215]. In order to fulfill this promise, sorafenib, has beenevealed as the first agent to show favorable overall survivaln patients with advanced HCC. Sorafenib is an oral multi-inase inhibitor that inhibits VEGFR1, VEGFR2, VEGFR3nd PDGFR-�, PDGFR-�, c-KIT, Raf-1 and BRAF. Earlyvidence of antitumor activity was observed from a phase
I study of 137 patients with advanced HCC: time-to-umor progression was 4.2 months and overall survival 9.2
onths [216]. Two subsequent randomized phase III stud-

lRk

able 3rognostic potential of serum markers in HCC.

ype of marker Poor prognosis

53 and related genes p53 mutation in plasma DNAenomic instability CSI, mutation in tumor suppressor gene, microselomerase activity Plasma telomerase activitydhesion molecules High sICAM-1 levelsCM degradation High levels of plasma MMP-9ngiogenesis related High levels of VEGF, b-FGFrowth factors High levels of TGF-�, TSGF, leptinenetic biomarkers High levels of AFP mRNA, IGF-II, albumin mRumor marker AFP, GPC-3, GOPH2nzymes/isoenzymes High levels of GGT, AFU, DCP

nflammatory marker High levels of CRPicroRNA miR500

NA: deoxy ribonucleic acid, CSI: chromosomal instability LOH: loss of heterozular matrix, MMP: matrix metalloproteases, VEGF: vascular endothelial growthransforming growth factor beta, TSGF: tumor specific growth factor, AFP mRN

RNA, hTERT mRNA: human telomerase reverse transcriptase mRNA, AFP: alphaamma-glutamyl transferase, AFU: alpha fucosidase, DCP: des-gamma-carboxypro

xide synthase, b-, FGF: basic fibroblast growth factor, IL-8: interleukin-8,owth factor receptor, GGT mRNA: gamma-glutamyl transferase mRNA,

es have also demonstrated improved overall survival andime-to-tumor progression [217,218]. Currently there areandomized studies evaluating the safety and efficacy oforafenib after curative resection [219]. Few recent studiesave shown a potential benefit of sorafenib in reducing thencidence of HCC recurrence and in extending disease-freend overall survival for high-risk liver transplant recipients220,221]. Another potential targets are abnormal growthactors and receptors, which stimulate several intracellu-
ar signal transduction pathways. Two major pathways, theas/Raf/MEK/MAPK and PI3 K/Akt/mTOR pathways, arenown to be involved in the oncogene addiction of HCC
Good prognosis

–atellite alterations, LOH in plasma DNA –

–––Low IL-8 levels–

NA, hTERT mRNA –––––

ygosity, sICAM: serum intercellular adhesion molecule-1, ECM: extracel-factor, b-FGF: basic fibroblast growth factor, IL-8: interleukin-8, TGF-�:A: alpha-fetoprotein mRNA, IGF-II mRNA: insulin-like growth factor-IIfetoprotein, GPC-3: glypican-3, GOLPH2: Golgi phosphoprotein 2, GGT:thrombin, CRP: c-reactive protein, miR: microRNA.

1 Oncolo

[tPikuP7a[thtomnc

1

hbtthma

C

R

Do

D

nO

R


222]. Regorafenib potently inhibits the angiopoietin recep-or Tie2 as well as potently inhibiting VEGFR2, VEGFR3,DGFR-beta, c-kit, Flt-3, and Raf kinases, and has advanced

nto clinical phase II trials for HCC therapy [223]. A MEKinase inhibitor, AZD6244 (ARRY-142886), has been eval-ated for HCC in clinical trials. In the other pathway,I3K/Akt/mTOR, the mTOR inhibitors temsirolimus (CCI-79), everolimus (RAD001), and AZD8055 are now underctive investigation in clinical trials for patients with HCC224]. These recent developments underscore the need forhorough and well-designed, randomized studies of thisighly heterogeneous disease. These approaches should leado a better selection of patients for targeted therapy basedn biomarkers, and should provide critical insight into theechanisms of resistance, thus facilitating the discovery of

ew targets. In turn, this may finally allow us optimize theurrent therapies for this dreadful disease.

0. Conclusion

Several molecular markers with prognostic significanceave been identified in HCC. However, most of them haveeen studied retrospectively. Efforts should be directedowards prospective clinical trials in evaluating the prognos-ic significance of these markers. These molecules not onlyelp in prediction of prognosis for patients with HCC, butay also assist in deciding appropriate modality of therapy

nd represent novel targets for therapeutic agents.

onflict of interest statement

None.

eviewers

Dr. Ann Lii Cheng, National Taiwan University Hospital,epartment of Internal Medicine and Department of Oncol-gy, No. 7, Chung-Shan South Road, Taipei, Taiwan

Dr. Linda Leung, The Chinese University of Hong Kong,epartment of Clinical Oncology, Shatin, Hong KongDr. Stephen L. Chan, Prince of Wales Hospital, the Chi-

ese University of Hong Kong, Department of Clinicalncology, 30-32 Ngan Shing Street, Shatin, Hong Kong

eferences

[1] Marrero JA. Hepatocellular carcinoma. Curr Opin Gastroenterol2006;22(3):248–53.

[2] Motola-Kuba D, Zamora-Valdes D, Uribe M, Mendez-Sanchez
N. Hepatocellular carcinoma. An overview. Ann Hepatol2006;5(1):16–24.
[3] Blum HE. Hepatocellular carcinoma: therapy and prevention. WorldJ Gastroenterol 2005;11(47):7391–400.

gy/Hematology 82 (2012) 116–140

[4] Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005. CA CancerJ Clin 2005;55(1):10–30.

[5] El-Serag HB. Epidemiology of hepatocellular carcinoma. Clin LiverDis 2001;5:87–107.

[6] Bismuth H, Majno PE, Adam R. Liver transplantation for hepatocel-lular carcinoma. Semin Liver Dis 1999;19(3):311–22.

[7] Zavaglia C, De Carlis L, Alberti AB, et al. Predictors of long-termsurvival after liver transplantation for hepatocellular carcinoma. AmJ Gastroenterol 2005;100(12):2708–16.

[8] Zimmerman MA, Ghobrial RM, Tong MJ, et al. Recurrence ofhepatocellular carcinoma following liver transplantation. A Reviewof preoperative and postoperative prognostic indicators. Arch Surg2008;143(2):182–8.

[9] Lai EC, Ng IO, Ng MM, et al. Long-term results of resection for largehepatocellular carcinoma: a multivariate analysis of clinicopatholog-ical features. Hepatology 1990;11(5):815–8.

[10] Shirabe K, Kanematsu T, Matsumata T, Adachi E, Akazawa K, Sug-imachi K. Factors linked to early recurrence of small hepatocellularcarcinoma after hepatectomy: univariate and multivariate analyses.Hepatology 1991;14(5):802–5.

[11] Jwo SC, Chiu JH, Chau GY, Loong CC, Lui WY. Risk factors linkedto tumor recurrence of human hepatocellular carcinoma after hepaticresection. Hepatology 1992;16(6):1367–71.

[12] Ikeda K, Saitoh S, Tsubota A, et al. Risk factors for tumor recurrenceand prognosis after curative resection of hepatocellular carcinoma.Cancer 1993;71(1):19–25.

[13] Giannelli G, Antonaci S. Novel concepts in hepatocellular carcinoma:from molecular research to clinical practice. J Clin Gastroenterol2006;40(9):842–6.

[14] Cha C, Dematteo RP. Molecular mechanisms in hepatocellu-lar carcinoma development. Best Pract Res Clin Gastroenterol2005;19(1):25–37.

[15] Farazi PA, DePinho RA. Hepatocellular carcinoma pathogenesis:from genes to environment. Nat Rev Cancer 2006;6(9):674–87.

[16] Mann CD, Neal CP, Garcea G, Manson MM, Dennison AR, BerryDP. Prognostic molecular markers in hepatocellular carcinoma: asystematic review. Eur J Cancer 2007;43(6):979–92.

[17] Qin LX, Tang ZY. The prognostic molecular markers in hepatocellularcarcinoma. World J Gastroenterol 2002;8(3):385–92.

[18] Travali S, Ku DH, Rizzo MG, Ottavio L, Baserga R, Calabretta B.Structure of the human gene for the proliferating cell nuclear antigen.J Biol Chem 1989;264(13):7466–72.

[19] Korkolopoulou P, Patsouris E, Pangalis G, et al. A comparative assess-ment of proliferating cell nuclear antigen, c-myc p62, and nucleolarorganizer region staining in non-Hodgkin’s lymphomas: a histo-chemical and immunohistochemical study of 200 cases. Hum Pathol1993;24(4):371–7.

[20] Ng IO, Lai EC, Fan ST, Ng M, Chan AS, So MK. Prognostic signifi-cance of proliferating cell nuclear antigen expression in hepatocellularcarcinoma. Cancer 1994;73(9):2268–74.

[21] Hino N, Higashi T, Nouso K, et al. Proliferating cell nuclearantigen and grade of malignancy in small hepatocellular carci-noma – evaluation in US-guided specimens. Hepatogastroenterology1997;44(13):245–50.

[22] Lin GY, Chen ZL, Lu CM, Li Y, Ping XJ, Huang R. Immunohis-tochemical study on p53, H-rasp21, c-erbB-2 protein and PCNAexpression in HCC tissues of Han and minority ethnic patients. WorldJ Gastroenterol 2000;6(2):234–8.

[23] Stroescu C, Dragnea A, Ivanov B, et al. Expression of p53, Bcl-2,VEGF, Ki67 and PCNA and prognostic significance in hepatocellularcarcinoma. J Gastrointest Liver Dis 2008;17(4):411–7.

[24] Osada S, Saji S, Kuno T. Clinical significance of combination studyof apoptotic factors and proliferating cell nuclear antigen in esti-
mating the prognosis of hepatocellular carcinoma. J Surg Oncol2004;85(1):48–54.
[25] Nan KJ, Guo H, Ruan ZP, Jing Z, Liu SX. Expression of p57(kip2) and its relationship with clinicopathology, PCNA and p53 in

Oncolo
primary hepatocellular carcinoma. World J Gastroenterol 2005;11(8):1237–40.

[26] Bullwinkel J, Baron-Luhr B, Ludemann A, Wohlenberg C, GerdesJ, Scholzen T. Ki-67 protein is associated with ribosomal RNAtranscription in quiescent and proliferating cells. J Cell Physiol2006;206(3):624–35.

[27] Scholzen T, Gerdes J. The Ki-67 protein: from the known and theunknown. J Cell Physiol 2000;182(3):311–22.

[28] Cui J, Dong B-W, Liang P, Yu X-L, Yu D-J. Effect of c-myc, Ki-67, MMP-2 and VEGF expression on prognosis of hepatocellularcarcinoma patients undergoing tumor resection. World J Gastroenterol2004;10(10):1533–6.

[29] Guzman G, Alagiozian-Angelova V, Layden-Almer JE, et al. p53,Ki-67, and serum alpha feto-protein as predictors of hepatocellu-lar carcinoma recurrence in liver transplant patients. Mod Pathol2005;18(11):1498–503.

[30] Fiorentino M, Altimari A, Ravaioli M, et al. Predictive valueof biological markers for hepatocellular carcinoma patientstreated with orthotopic liver transplantation. Clin Cancer Res2004;10(5):1789–95.

[31] Behrens P, Brinkmann U, Wellmann A. CSE1L/CAS: its role in pro-liferation and apoptosis. Apoptosis 2003;8(1):39–44.

[32] Brinkmann U. CAS, the human homologue of the yeast chromosome-segregation gene CSE1, in proliferation, apoptosis, and cancer. Am JHum Genet 1998;62(3):509–13.

[33] Wellmann A, Flemming P, Behrens P, et al. High expression of theproliferation and apoptosis associated CSE1L/CAS gene in hepatitisand liver neoplasms: correlation with tumor progression. Int J MolMed 2001;7(5):489–94.

[34] Shiraki K, Fujikawa K, Sugimoto K, et al. Cellular apoptosis suscep-tibility protein and proliferation in human hepatocellular carcinoma.Int J Mol Med 2006;18(1):77–81.

[35] Liao CF, Luo SF, Li LT, Lin CY, Chen YC, Jiang MC. CSE1L/CAS,the cellular apoptosis susceptibility protein, enhances invasion andmetastasis but not proliferation of cancer cells. J Exp Clin CancerRes 2008;27:15.

[36] Ikeguchi M, Sato N, Hirooka Y, Kaibara N. Computerized nuclearmorphometry of hepatocellular carcinoma and its relation to prolifer-ative activity. J Surg Oncol 1998;68(4):225–30.

[37] Veltri RW, Partin AW, Miller MC. Quantitative nuclear grade (QNG):a new image analysis-based biomarker of clinically relevant nuclearstructure alterations. J Cell Biochem Suppl 2000;Suppl. 35:151–7.

[38] Blaszyk H, Hartmann A, Cunningham JM, et al. A prospective trial ofmidwest breast cancer patients: a p53 gene mutation is the most impor-tant predictor of adverse outcome. Int J Cancer 2000;89(1):32–8.

[39] Kahlenberg MS, Stoler DL, Rodriguez-Bigas MA, et al. p53 tumorsuppressor gene mutations predict decreased survival of patients withsporadic colorectal carcinoma. Cancer 2000;88(8):1814–9.

[40] Ireland AP, Shibata DK, Chandrasoma P, Lord RV, Peters JH,DeMeester TR. Clinical significance of p53 mutations in adenocarci-noma of the esophagus and cardia. Ann Surg 2000;231(2):179–87.

[41] Tamas L, Kraxner H, Mechtler L, et al. Prognostic significance ofP53 histochemistry and DNA histogram parameters in head and neckmalignancies. Anticancer Res 2000;20(5C):4031–7.

[42] Mitsudomi T, Hamajima N, Ogawa M, Takahashi T. Prognostic signif-icance of p53 alterations in patients with non-small cell lung cancer:a meta-analysis. Clin Cancer Res 2000;6(10):4055–63.

[43] Shahin MS, Hughes JH, Sood AK, Buller RE. The prognostic signifi-cance of p53 tumor suppressor gene alterations in ovarian carcinoma.Cancer 2000;89(9):2006–17.

[44] Soini Y, Virkajarvi N, Lehto VP, Paakko P. Hepatocellular carcino-mas with a high proliferation index and a low degree of apoptosisand necrosis are associated with a shortened survival. Br J Cancer
1996;73(9):1025–30.
[45] Sheen IS, Jeng KS, Wu JY. Is p53 gene mutation an indicatior ofthe biological behaviors of recurrence of hepatocellular carcinoma?World J Gastroenterol 2003;9(6):1202–7.

gy/Hematology 82 (2012) 116–140 135

[46] Itoh T, Hayashi Y, Kanamaru T, et al. Clinical significance ofurokinase-type plasminogen activator activity in hepatocellular car-cinoma. J Gastroenterol Hepatol 2000;15(4):422–30.

[47] Jeng KS, Sheen IS, Chen BF, Wu JY. Is the p53 gene mutation ofprognostic value in hepatocellular carcinoma after resection? ArchSurg 2000;135(11):1329–33.

[48] Shiota G, Kishimoto Y, Suyama A, et al. Prognostic significance ofserum anti-p53 antibody in patients with hepatocellular carcinoma. JHepatol 1997;27(4):661–8.

[49] Qin LX, Tang ZY, Ma ZC, et al. P53 immunohistochemical scoring:an independent prognostic marker for patients after hepatocellularcarcinoma resection. World J Gastroenterol 2002;8(3):459–63.

[50] Sugo H, Takamori S, Kojima K, Beppu T, Futagawa S. The signifi-cance of p53 mutations as an indicator of the biological behavior ofrecurrent hepatocellular carcinomas. Surg Today 1999;29(9):849–55.

[51] Lowe SW, Bodis S, McClatchey A, et al. p53 status and the efficacyof cancer therapy in vivo. Science 1994;266(5186):807–10.

[52] Endo K, Ueda T, Ohta T, Terada T. Protein expression of MDM2 andits clinicopathological relationships in human hepatocellular carci-noma. Liver 2000;20(3):209–15.

[53] Tannapfel A, Wasner M, Krause K, et al. Expression of p73 and itsrelation to histopathology and prognosis in hepatocellular carcinoma.J Natl Cancer Inst 1999;91(13):1154–8.

[54] Katoh H, Shibata T, Kokubu A, et al. Genetic profile of hepatocellularcarcinoma revealed by array-based comparative genomic hybridiza-tion: identification of genetic indicators to predict patient outcome. JHepatol 2005;43(5):863–74.

[55] Kusano N, Okita K, Shirahashi H, et al. Chromosomal imbal-ances detected by comparative genomic hybridization are associatedwith outcome of patients with hepatocellular carcinoma. Cancer2002;94(3):746–51.

[56] Itano O, Ueda M, Kikuchi K, et al. A new predictive factor for hep-atocellular carcinoma based on two-dimensional electrophoresis ofgenomic DNA. Oncogene 2000;19(13):1676–83.

[57] Knudson AG. Genetics and etiology of human cancer. Adv HumGenet 1977;8:1–66.

[58] Marsh JW, Finkelstein SD, Demetris AJ, et al. Genotyping ofhepatocellular carcinoma in liver transplant recipients adds predic-tive power for determining recurrence-free survival. Liver Transpl2003;9(7):664–71.

[59] Macdonald GA, Greenson JK, Saito K, Cherian SP, Appelman HD,Boland CR. Microsatellite instability and loss of heterozygosity atDNA mismatch repair gene loci occurs during hepatic carcinogenesis.Hepatology 1998;28(1):90–7.

[60] Yamamoto H, Itoh F, Fukushima H, et al. Infrequent widespreadmicrosatellite instability in hepatocellular carcinomas. Int J Oncol2000;16(3):543–7.

[61] Chiappini F, Gross-Goupil M, Saffroy R, et al. Microsatelliteinstability mutator phenotype in hepatocellular carcinoma in non-alcoholic and non-virally infected normal livers. Carcinogenesis2004;25(4):541–7.

[62] Chao Y, Shih YL, Chiu JH, et al. Overexpression of cyclin A butnot Skp 2 correlates with the tumor relapse of human hepatocellularcarcinoma. Cancer Res 1998;58(5):985–90.

[63] Ohashi R, Gao C, Miyazaki M, et al. Enhanced expression of cyclinE and cyclin A in human hepatocellular carcinomas. Anticancer Res2001;21(1B):657–62.

[64] Tannapfel A, Anhalt K, Hausermann P, et al. Identification of novelproteins associated with hepatocellular carcinomas using proteinmicroarrays. J Pathol 2003;201(2):238–49.

[65] Li X, Hui A-M, Sun L, et al. p16INK4A hypermethylation is associ-ated with hepatitis virus infection, age, and gender in hepatocellularcarcinoma. Clin Cancer Res 2004;10(22):7484–9.

[66] Weihrauch M, Benicke M, Lehnert G, Wittekind C, Wrbitzky R, Tan-napfel A. Frequent k-ras-2 mutations and p16(INK4A)methylation inhepatocellular carcinomas in workers exposed to vinyl chloride. Br JCancer 2001;84(7):982–9.

1 Oncolo
[67] Matsuda Y. Molecular mechanism underlying the functional loss ofcyclindependent kinase inhibitors p16 and p27 in hepatocellular car-cinoma. World J Gastroenterol 2008;14(11):1734–40.

[68] Wong IH, Lo YM, Yeo W, Lau WY, Johnson PJ. Frequent p15 pro-moter methylation in tumor and peripheral blood from hepatocellularcarcinoma patients. Clin Cancer Res 2000;6(9):3516–21.

[69] Morishita A, Masaki T, Yoshiji H, et al. Reduced expression ofcell cycle regulator p18(INK4C) in human hepatocellular carcinoma.Hepatology 2004;40(3):677–86.

[70] Fiorentino M, Altimari A, D’Errico A, et al. Acquired expression ofp27 is a favorable prognostic indicator in patients with hepatocellularcarcinoma. Clin Cancer Res 2000;6(10):3966–72.

[71] Ito Y, Matsuura N, Sakon M, et al. Expression and prognostic roles ofthe G1-S modulators in hepatocellular carcinoma: p27 independentlypredicts the recurrence. Hepatology 1999;30(1):90–9.

[72] Tannapfel A, Grund D, Katalinic A, et al. Decreased expression ofp27 protein is associated with advanced tumor stage in hepatocellularcarcinoma. Int J Cancer 2000;89(4):350–5.

[73] Cui J, Dong BW, Liang P, Yu XL, Yu DJ. Construction and clinicalsignificance of a predictive system for prognosis of hepatocellularcarcinoma. World J Gastroenterol 2005;11(20):3027–33.

[74] Wong N, Yeo W, Wong W, et al. TOP2A overexpression in hepato-cellular carcinoma correlates with early age onset, shorter patientssurvival and chemoresistance. Int J Cancer 2009;124(3):644–52.

[75] Ito Y, Miyoshi E, Takeda T, et al. Expression and possible role of ets-1in hepatocellular carcinoma. Am J Clin Pathol 2000;114(5):719–25.

[76] Garcia EJ, Lawson D, Cotsonis G, Cohen C. Hepatocellular carcinomaand markers of apoptosis (bcl-2, bax, bcl-x): prognostic significance.Appl Immunohistochem Mol Morphol 2002;10(3):210–7.

[77] Watanabe J, Kushihata F, Honda K, et al. Prognostic signif-icance of Bcl-xL in human hepatocellular carcinoma. Surgery2004;135(6):604–12.

[78] Ito Y, Monden M, Takeda T, et al. The status of Fas and Fas ligandexpression can predict recurrence of hepatocellular carcinoma. Br JCancer 2000;82(6):1211–7.

[79] Gao Q, Wang XY, Qiu SJ, et al. Overexpression of PD-L1 significantlyassociates with tumor aggressiveness and postoperative recurrencein human hepatocellular carcinoma. Clin Cancer Res 2009;15(3):971–9.

[80] Fields AC, Cotsonis G, Sexton D, Santoianni R, Cohen C. Sur-vivin expression in hepatocellular carcinoma: correlation withproliferation, prognostic parameters, and outcome. Mod Pathol2004;17(11):1378–85.

[81] Ikeguchi M, Ueda T, Sakatani T, Hirooka Y, Kaibara N. Expressionof survivin messenger RNA correlates with poor prognosis in patientswith hepatocellular carcinoma. Diagn Mol Pathol 2002;11(1):33–40.

[82] Chen C-J, Kyo S, Liu Y-C, et al. Modulation of human telomerasereverse transcriptase in hepatocellular carcinoma. World J Gastroen-terol 2004;10(5):638–42.

[83] Kishimoto K, Fujimoto J, Takeuchi M, Yamamoto H, Ueki T,Okamoto E. Telomerase activity in hepatocellular carcinoma andadjacent liver tissues. J Surg Oncol 1998;69(3):119–24.

[84] Kobayashi T, Kubota K, Takayama T, Makuuchi M. Telomerase activ-ity as a predictive marker for recurrence of hepatocellular carcinomaafter hepatectomy. Am J Surg 2001;181(3):284–8.

[85] Tatsuma T, Goto S, Kitano S, Lin YC, Lee CM, Chen CL. Telomeraseactivity in peripheral blood for diagnosis of hepatoma. J GastroenterolHepatol 2000;15(9):1064–70.

[86] Nelson WJ, Nusse R. Convergence of Wnt, beta-catenin, and cadherinpathways. Science 2004;303(5663):1483–7.

[87] Aravalli RN, Steer CJ, Cressman EN. Molecular mechanisms of hep-atocellular carcinoma. Hepatology 2008;48(6):2047–63.

[88] Endo K, Ueda T, Ueyama J, Ohta T, Terada T. Immunoreactive
E-cadherin, alpha-catenin, beta-catenin, and gamma-catenin pro-teins in hepatocellular carcinoma: relationships with tumor grade,clinicopathologic parameters, and patients’ survival. Hum Pathol2000;31(5):558–65.
gy/Hematology 82 (2012) 116–140

[89] Inagawa S, Itabashi M, Adachi S, et al. Expression and prognos-tic roles of beta-catenin in hepatocellular carcinoma: correlationwith tumor progression and postoperative survival. Clin Cancer Res2002;8(2):450–6.

[90] Soyama A, Eguchi S, Takatsuki M, et al. Significance of the serumlevel of soluble E-cadherin in patients with HCC. Hepatogastroen-terology 2008;55(85):1390–3.

[91] Hsu HC, Jeng YM, Mao TL, Chu JS, Lai PL, Peng SY. Beta-cateninmutations are associated with a subset of low-stage hepatocellularcarcinoma negative for hepatitis B virus and with favorable prognosis.Am J Pathol 2000;157(3):763–70.

[92] Wong CM, Fan ST, Ng IO. Beta-catenin mutation and overexpres-sion in hepatocellular carcinoma: clinicopathologic and prognosticsignificance. Cancer 2001;92(1):136–45.

[93] Lee HH, Uen YH, Tian YF, et al. Wnt-1 protein as a prognosticbiomarker for hepatitis B-related and hepatitis C-related hepatocel-lular carcinoma after surgery. Cancer Epidemiol Biomarkers Prev2009;18(5):1562–9.

[94] Zhai B, Yan HX, Liu SQ, Chen L, Wu MC, Wang HY. Reduced expres-sion of E-cadherin/catenin complex in hepatocellular carcinomas.World J Gastroenterol 2008;14(37):5665–73.

[95] Korita PV, Wakai T, Shirai Y, et al. Overexpression of osteo-pontin independently correlates with vascular invasion and poorprognosis in patients with hepatocellular carcinoma. Hum Pathol2008;39(12):1777–83.

[96] Goodison S, Urquidi V, Tarin D. CD44 cell adhesion molecules. MolPathol 1999;52(4):189–96.

[97] Endo K, Terada T. Protein expression of CD44 (standard and vari-ant isoforms) in hepatocellular carcinoma: relationships with tumorgrade, clinicopathologic parameters, p53 expression, and patient sur-vival. J Hepatol 2000;32(1):78–84.

[98] Yang GH, Fan J, Xu Y, et al. Osteopontin combined with CD44, a novelprognostic biomarker for patients with hepatocellular carcinomaundergoing curative resection. Oncologist 2008;13(11):1155–65.

[99] Mei MH, Xu J, Shi QF, Yang JH, Chen Q, Qin LL. Clinicalsignificance of serum intercellular adhesion molecule-1 detectionin patients with hepatocellular carcinoma. World J Gastroenterol2000;6(3):408–10.

[100] Fritz G, Brachetti C, Bahlmann F, Schmidt M, Kaina B. Rho GTPasesin human breast tumours: expression and mutation analyses and cor-relation with clinical parameters. Br J Cancer 2002;87(6):635–44.

[101] Suwa H, Ohshio G, Imamura T, et al. Overexpression of the rhoC genecorrelates with progression of ductal adenocarcinoma of the pancreas.Br J Cancer 1998;77(1):147–52.

[102] Horiuchi A, Imai T, Wang C, et al. Up-regulation of small GTPases,RhoA and RhoC, is associated with tumor progression in ovariancarcinoma. Lab Invest 2003;83(6):861–70.

[103] Wang W, Yang LY, Huang GW, et al. Genomic analysis reveals RhoCas a potential marker in hepatocellular carcinoma with poor prognosis.Br J Cancer 2004;90(12):2349–55.

[104] Xiaorong L, Wei W, Liyuan Q, Kaiyan Y. Underexpression ofdeleted in liver cancer 2 (DLC2) is associated with overexpression ofRhoA and poor prognosis in hepatocellular carcinoma. BMC Cancer2008;8:205.

[105] Moseley JB, Bartolini F, Okada K, Wen Y, Gundersen GG, GoodeBL. Regulated binding of adenomatous polyposis coli protein to actin.J Biol Chem 2007;282(17):12661–8.

[106] Orimo T, Ojima H, Hiraoka N, et al. Proteomic profiling reveals theprognostic value of adenomatous polyposis coli-end-binding protein1 in hepatocellular carcinoma. Hepatology 2008;48(6):1851–63.

[107] Theret N, Musso O, Turlin B, et al. Increased extracellular matrixremodeling is associated with tumor progression in human hepato-cellular carcinomas. Hepatology 2001;34(1):82–8.

[108] Yamamoto H, Itoh F, Adachi Y, et al. Relation of enhancedsecretion of active matrix metalloproteinases with tumor spreadin human hepatocellular carcinoma. Gastroenterology 1997;112(4):1290–6.

Oncolo
[109] Korn WM. Moving toward an understanding of the metastatic processin hepatocellular carcinoma. World J Gastroenterol 2001;7(6):777–8.

[110] Gorrin Rivas MJ, Arii S, Furutani M, et al. Expression of humanmacrophage metalloelastase gene in hepatocellular carcinoma: cor-relation with angiostatin generation and its clinical significance.Hepatology 1998;28(4):986–93.

[111] Itoh T, Shiro T, Seki T, et al. Relationship between p53 overexpressionand the proliferative activity in hepatocellular carcinoma. Int J MolMed 2000;6(2):137–42.

[112] Zheng Q, Tang ZY, Xue Q, Shi DR, Song HY, Tang HB. Invasion andmetastasis of hepatocellular carcinoma in relation to urokinase-typeplasminogen activator, its receptor and inhibitor. J Cancer Res ClinOncol 2000;126(11):641–6.

[113] Jiang YF, Yang ZH, Hu JQ. Recurrence or metastasis ofHCC:predictors, early detection and experimental antiangiogenictherapy. World J Gastroenterol 2000;6(1):61–5.

[114] Morinaga S, Imada T, Shimizu A, et al. Angiogenesis in hepatocellularcarcinoma as evaluated by alpha smooth muscle actin immunohisto-chemistry. Hepatogastroenterology 2001;48(37):224–8.

[115] Poon RT, Ng IO, Lau C, et al. Tumor microvessel density as a pre-dictor of recurrence after resection of hepatocellular carcinoma: aprospective study. J Clin Oncol 2002;20(7):1775–85.

[116] Ho JW, Poon RT, Sun CK, Xue WC, Fan ST. Clinicopathologicaland prognostic implications of endoglin (CD105) expression in hep-atocellular carcinoma and its adjacent non-tumorous liver. World JGastroenterol 2005;11(2):176–81.

[117] El-Assal ON, Yamanoi A, Soda Y, et al. Clinical significance ofmicrovessel density and vascular endothelial growth factor expressionin hepatocellular carcinoma and surrounding liver: possible involve-ment of vascular endothelial growth factor in the angiogenesis ofcirrhotic liver. Hepatology 1998;27(6):1554–62.

[118] Jeng KS, Sheen IS, Wang YC, et al. Is the vascular endothelial growthfactor messenger RNA expression in resectable hepatocellular car-cinoma of prognostic value after resection? World J Gastroenterol2004;10(5):676–81.

[119] Poon RT, Ho JW, Tong CS, Lau C, Ng IO, Fan ST. Prognos-tic significance of serum vascular endothelial growth factor andendostatin in patients with hepatocellular carcinoma. Br J Surg2004;91(10):1354–60.

[120] Chao Y, Li CP, Chau GY, et al. Prognostic significance of vas-cular endothelial growth factor, basic fibroblast growth factor, andangiogenin in patients with resectable hepatocellular carcinoma aftersurgery. Ann Surg Oncol 2003;10(4):355–62.

[121] Jia JB, Zhuang PY, Sun HC, et al. Protein expression profiling of vas-cular endothelial growth factor and its receptors identifies subclassesof hepatocellular carcinoma and predicts survival. J Cancer Res ClinOncol 2009;135(6):847–54.

[122] Zhou J, Tang ZY, Fan J, et al. Expression of platelet-derived endothe-lial cell growth factor and vascular endothelial growth factor inhepatocellular carcinoma and portal vein tumor thrombus. J CancerRes Clin Oncol 2000;126(1):57–61.

[123] Ezaki T, Ikegami T, Maeda T, et al. Prognostic value of thymidinephosphorylase activity in liver tissue adjacent to hepatocellular carci-noma. Int J Clin Oncol 2005;10(3):171–6.

[124] Hu J, Xu Y, Shen ZZ, et al. High expressions of vascular endothe-lial growth factor and platelet-derived endothelial cell growth factorpredict poor prognosis in alpha-fetoprotein-negative hepatocellularcarcinoma patients after curative resection. J Cancer Res Clin Oncol2009;135(10):1359–67.

[125] Wada H, Nagano H, Yamamoto H, et al. Expression pattern of angio-genic factors and prognosis after hepatic resection in hepatocellularcarcinoma: importance of angiopoietin-2 and hypoxia-induced factor-1 alpha. Liver Int 2006;26(4):414–23.

[126] Ikeguchi M, Ueta T, Yamane Y, Hirooka Y, Kaibara N. Induciblenitric oxide synthase and survivin messenger RNA expres-sion in hepatocellular carcinoma. Clin Cancer Res 2002;8(10):3131–6.

gy/Hematology 82 (2012) 116–140 137

[127] Rahman MA, Dhar DK, Yamaguchi E, et al. Coexpression of induciblenitric oxide synthase and COX-2 in hepatocellular carcinoma andsurrounding liver: possible involvement of COX-2 in the angiogen-esis of hepatitis C virus-positive cases. Clin Cancer Res 2001;7(5):1325–32.

[128] Poon RT, Ng IO, Lau C, Yu WC, Fan ST, Wong J. Correlation of serumbasic fibroblast growth factor levels with clinicopathologic featuresand postoperative recurrence in hepatocellular carcinoma. Am J Surg2001;182(3):298–304.

[129] Kaido T, Oe H, Yoshikawa A, Mori A, Arii S, Imamura M.Tissue factor is a useful prognostic factor of recurrence in hep-atocellular carcinoma in 5-year survivors. Hepatogastroenterology2005;52(65):1383–7.

[130] Hu TH, Huang CC, Wu CL, et al. Increased endostatin/collagen XVIIIexpression correlates with elevated VEGF level and poor prognosisin hepatocellular carcinoma. Mod Pathol 2005;18(5):663–72.

[131] Strieter RM, Kunkel SL, Elner VM, et al. Interleukin-8. Acorneal factor that induces neovascularization. Am J Pathol1992;141(6):1279–84.

[132] Luca M, Huang S, Gershenwald JE, Singh RK, Reich R, Bar-Eli M.Expression of interleukin-8 by human melanoma cells up-regulatesMMP-2 activity and increases tumor growth and metastasis. Am JPathol 1997;151(4):1105–13.

[133] Ren Y, Poon RT, Tsui HT, et al. Interleukin-8 serum levels in patientswith hepatocellular carcinoma: correlations with clinicopathologicalfeatures and prognosis. Clin Cancer Res 2003;9(16 Pt 1):5996–6001.

[134] Akiba J, Yano H, Ogasawara S, Higaki K, Kojiro M. Expression andfunction of interleukin-8 in human hepatocellular carcinoma. Int JOncol 2001;18(2):257–64.

[135] Kubo F, Ueno S, Hiwatashi K, et al. Interleukin 8 in human hepato-cellular carcinoma correlates with cancer cell invasion of vessels butnot with tumor angiogenesis. Ann Surg Oncol 2005;12(10):800–7.

[136] Mitsuhashi N, Shimizu H, Ohtsuka M, et al. Angiopoietins and Tie-2expression in angiogenesis and proliferation of human hepatocellularcarcinoma. Hepatology 2003;37(5):1105–13.

[137] Okumoto K, Hattori E, Tamura K, et al. Possible contribution of cir-culating transforming growth factor-beta1 to immunity and prognosisin unresectable hepatocellular carcinoma. Liver Int 2004;24(1):21–8.

[138] Ikeguchi M, Iwamoto A, Taniguchi K, Katano K, Hirooka Y. The geneexpression level of transforming growth factor-beta (TGF-beta) as abiological prognostic marker of hepatocellular carcinoma. J Exp ClinCancer Res 2005;24(3):415–21.

[139] Song BC, Chung YH, Kim JA, et al. Transforming growth factor-beta1 as a useful serologic marker of small hepatocellular carcinoma.Cancer 2002;94(1):175–80.

[140] Zhou L, Liu J, Luo F. Serum tumor markers for detection of hepato-cellular carcinoma. World J Gastroenterol 2006;12(8):1175–81.

[141] Tsai JF, Jeng JE, Chuang LY, et al. Elevated urinary transform-ing growth factor-beta1 level as a tumour marker and predictor ofpoor survival in cirrhotic hepatocellular carcinoma. Br J Cancer1997;76(2):244–50.

[142] Ito Y, Takeda T, Sakon M, et al. Expression and clinical significanceof erb-B receptor family in hepatocellular carcinoma. Br J Cancer2001;84(10):1377–83.

[143] Osada S, Kanematsu M, Imai H, Goshima S. Clinical significanceof serum HGF and c-Met expression in tumor tissue for evaluationof properties and treatment of hepatocellular carcinoma. Hepatogas-troenterology 2008;55(82–83):544–9.

[144] Soresi M, Magliarisi C, Campagna P, et al. Usefulness of alpha-fetoprotein in the diagnosis of hepatocellular carcinoma. AnticancerRes 2003;23(2C):1747–53.

[145] Tangkijvanich P, Anukulkarnkusol N, Suwangool P, et al. Clinicalcharacteristics and prognosis of hepatocellular carcinoma: analy-
sis based on serum alpha-fetoprotein levels. J Clin Gastroenterol2000;31(4):302–8.
[146] Fujioka M, Nakashima Y, Nakashima O, Kojiro M. Immunohistologicstudy on the expressions of alpha-fetoprotein and protein induced

1 Oncolo
by vitamin K absence or antagonist II in surgically resected smallhepatocellular carcinoma. Hepatology 2001;34(6):1128–34.

[147] Iida H, Honda M, Kawai HF, et al. Ephrin-A1 expression contributesto the malignant characteristics of {alpha}-fetoprotein producing hep-atocellular carcinoma. Gut 2005;54(6):843–51.

[148] Khien VV, Mao HV, Chinh TT, et al. Clinical evaluation of lentillectin-reactive alpha-fetoprotein-L3 in histology-proven hepatocellu-lar carcinoma. Int J Biol Markers 2001;16(2):105–11.

[149] Oka H, Saito A, Ito K, Kumada T, et al. Multicenter prospective anal-ysis of newly diagnosed hepatocellular carcinoma with respect tothe percentage of Lens culinaris agglutinin-reactive alpha-fetoprotein.J Gastroenterol Hepatol 2001;16(12):1378–83.

[150] Nassar A, Cohen C, Siddiqui MT. Utility of glypican-3 and survivinin differentiating hepatocellular carcinoma from benign and preneo-plastic hepatic lesions and metastatic carcinomas in liver fine-needleaspiration biopsies. Diagn Cytopathol 2009;37(9):629–35.

[151] Capurro M, Wanless IR, Sherman M, et al. Glypican-3: a novel serumand histochemical marker for hepatocellular carcinoma. Gastroen-terology 2003;125(1):89–97.

[152] Nakatsura T, Yoshitake Y, Senju S, et al. Glypican-3, overexpressedspecifically in human hepatocellular carcinoma, is a novel tumormarker. Biochem Biophys Res Commun 2003;306(1):16–25.

[153] Hippo Y, Watanabe K, Watanabe A, et al. Identification ofsoluble NH2-terminal fragment of glypican-3 as a serologi-cal marker for early-stage hepatocellular carcinoma. Cancer Res2004;64(7):2418–23.

[154] Shirakawa H, Suzuki H, Shimomura M, et al. Glypican-3 expressionis correlated with poor prognosis in hepatocellular carcinoma. CancerSci 2009;100(8):1403–7.

[155] Cui R, He J, Zhang F, et al. Diagnostic value of protein inducedby vitamin K absence (PIVKAII) and hepatoma-specific band ofserum gamma-glutamyl transferase (GGTII) as hepatocellular car-cinoma markers complementary to alpha-fetoprotein. Br J Cancer2003;88(12):1878–82.

[156] Tangkijvanich P, Tosukhowong P, Bunyongyod P, et al. Alpha-l-fucosidase as a serum marker of hepatocellular carcinoma in Thailand.Southeast Asian J Trop Med Public Health 1999;30(1):110–4.

[157] Ishizuka H, Nakayama T, Matsuoka S, et al. Prediction of the develop-ment of hepato-cellular-carcinoma in patients with liver cirrhosis bythe serial determinations of serum alpha-l-fucosidase activity. InternMed 1999;38(12):927–31.

[158] Suzuki M, Shiraha H, Fujikawa T, et al. Des-gamma-carboxy pro-thrombin is a potential autologous growth factor for hepatocellularcarcinoma. J Biol Chem 2005;280(8):6409–15.

[159] Cui R, Wang B, Ding H, Shen H, Li Y, Chen X. Usefulness ofdetermining a protein induced by vitamin K absence in detection ofhepatocellular carcinoma. Chin Med J (Engl) 2002;115(1):42–5.

[160] Marrero JA, Su GL, Wei W, et al. Des-gamma carboxypro-thrombin can differentiate hepatocellular carcinoma from nonma-lignant chronic liver disease in American patients. Hepatology2003;37(5):1114–21.

[161] Shimizu A, Shiraki K, Ito T, et al. Sequential fluctuation patternof serum des-gamma-carboxy prothrombin levels detected by high-sensitive electrochemiluminescence system as an early predictivemarker for hepatocellular carcinoma in patients with cirrhosis. IntJ Mol Med 2002;9(3):245–50.

[162] Hamamura K, Shiratori Y, Shiina S, et al. Unique clinical charac-teristics of patients with hepatocellular carcinoma who present withhigh plasma des-gamma-carboxy prothrombin and low serum alpha-fetoprotein. Cancer 2000;88(7):1557–64.

[163] Okuda H, Nakanishi T, Takatsu K, et al. Comparison of clini-copathological features of patients with hepatocellular carcinomaseropositive for alpha-fetoprotein alone and those seropositive for
des-gamma-carboxy prothrombin alone. J Gastroenterol Hepatol2001;16(11):1290–6.
[164] Kim HS, Park JW, Jang JS, et al. Prognostic values of alpha-fetoprotein and protein induced by vitamin K absence or antagonist-II

gy/Hematology 82 (2012) 116–140

in hepatitis B virus-related hepatocellular carcinoma: a prospectivestudy. J Clin Gastroenterol 2009;43(5):482–8.

[165] Tang W, Kokudo N, Sugawara Y, et al. Des-gamma-carboxyprothrombin expression in cancer and/or non-cancerliver tissues: association with survival of patients with resectablehepatocellular carcinoma. Oncol Rep 2005;13(1):25–30.

[166] Harino Y, Fujii M, Imura S, et al. The role of des-gamma-carboxyprothrombin expression in hepatocellular carcinoma. Hep-atogastroenterology 2008;55(85):1385–9.

[167] Kobayashi M, Ikeda K, Kawamura Y, et al. High serumdes-gamma-carboxy prothrombin level predicts poor prognosisafter radiofrequency ablation of hepatocellular carcinoma. Cancer2009;115(3):571–80.

[168] Marrero JA, Romano PR, Nikolaeva O, et al. GP73, a resident Golgiglycoprotein, is a novel serum marker for hepatocellular carcinoma.J Hepatol 2005;43(6):1007–12.

[169] Riener MO, Stenner F, Liewen H, et al. Golgi phosphopro-tein 2 (GOLPH2) expression in liver tumors and its value as aserum marker in hepatocellular carcinomas. Hepatology 2009;49(5):1602–9.

[170] Matsumura M, Niwa Y, Kato N, et al. Detection of alpha-fetoproteinmRNA, an indicator of hematogenous spreading hepatocellularcarcinoma, in the circulation: a possible predictor of metastatic hep-atocellular carcinoma. Hepatology 1994;20(6):1418–25.

[171] Ijichi M, Takayama T, Matsumura M, Shiratori Y, Omata M, Maku-uchi M. Alpha-fetoprotein mRNA in the circulation as a predictorof postsurgical recurrence of hepatocellular carcinoma: a prospectivestudy. Hepatology 2002;35(4):853–60.

[172] Jeng KS, Sheen IS, Tsai YC. Circulating messenger RNA of alpha-fetoprotein: a possible risk factor of recurrence after resection ofhepatocellular carcinoma. Arch Surg 2004;139(10):1055–60.

[173] Ding X, Yang LY, Huang GW, et al. Role of AFP mRNA expressionin peripheral blood as a predictor for postsurgical recurrence of hep-atocellular carcinoma: a systematic review and meta-analysis. WorldJ Gastroenterol 2005;11(17):2656–61.

[174] Sheen IS, Jeng KS, Tsai YC. Is the expression of gamma-glutamyltranspeptidase messenger RNA an indicator of biological behav-ior in recurrent hepatocellular carcinoma? World J Gastroenterol2003;9(3):468–73.

[175] Tsutsumi M, Sakamuro D, Takada A, Zang SC, Furukawa T,Taniguchi N. Detection of a unique gamma-glutamyl transpepti-dase messenger RNA species closely related to the developmentof hepatocellular carcinoma in humans: a new candidate for earlydiagnosis of hepatocellular carcinoma. Hepatology 1996;23(5):1093–7.

[176] Himoto T, Kuriyama S, Zhang JY, et al. Analyses of autoantibod-ies against tumor-associated antigens in patients with hepatocellularcarcinoma. Int J Oncol 2005;27(4):1079–85.

[177] Tsai JF, Jeng JE, Chuang LY, et al. Serum insulin-like growth factor-II as a serologic marker of small hepatocellular carcinoma. Scand JGastroenterol 2005;40(1):68–75.

[178] Cheung ST, Fan ST, Lee YT, et al. Albumin mRNA in plasma predictspost-transplant recurrence of patients with hepatocellular carcinoma.Transplantation 2008;85(1):81–7.

[179] Miura N, Maeda Y, Kanbe T, et al. Serum human telomerase reversetranscriptase messenger RNA as a novel tumor marker for hepatocel-lular carcinoma. Clin Cancer Res 2005;11(9):3205–9.

[180] Kong SY, Park JW, Kim JO, et al. Alpha-fetoprotein and humantelomerase reverse transcriptase mRNA levels in peripheral bloodof patients with hepatocellular carcinoma. J Cancer Res Clin Oncol2009;135(8):1091–8.

[181] Mou DC, Cai SL, Peng JR, et al. Evaluation of MAGE-1 and MAGE-3as tumour-specific markers to detect blood dissemination of hepato-
cellular carcinoma cells. Br J Cancer 2002;86(1):110–6.
[182] Yoon SK, Lim NK, Ha SA, et al. The human cervical cancer oncogeneprotein is a biomarker for human hepatocellular carcinoma. CancerRes 2004;64(15):5434–41.

Oncolo
[183] Cheung ST, Wong SY, Leung KL, et al. Granulin–epithelin precur-sor overexpression promotes growth and invasion of hepatocellularcarcinoma. Clin Cancer Res 2004;10(22):7629–36.

[184] Tricoli JV, Jacobson JW. MicroRNA: potential for cancer detection,diagnosis, and prognosis. Cancer Res 2007;67(10):4553–5.

[185] Yamamoto Y, Kosaka N, Tanaka M, et al. MicroRNA-500 as apotential diagnostic marker for hepatocellular carcinoma. Biomarkers2009;14(7):529–38.

[186] Xiong Y, Fang JH, Yun JP, et al. Effects of MicroRNA-29 on apo-ptosis, tumorigenicity, and prognosis of hepatocellular carcinoma.Hepatology 2009.

[187] Murakami Y, Yasuda T, Saigo K, et al. Comprehensive analysis ofmicroRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues. Oncogene 2006;25(17):2537–45.

[188] Budhu A, Jia HL, Forgues M, et al. Identification of metastasis-related microRNAs in hepatocellular carcinoma. Hepatology2008;47(3):897–907.

[189] Jiang J, Gusev Y, Aderca I, et al. Association of MicroRNA expressionin hepatocellular carcinomas with hepatitis infection, cirrhosis, andpatient survival. Clin Cancer Res 2008;14(2):419–27.

[190] Meng F, Henson R, Wehbe-Janek H, Ghoshal K, Jacob ST, PatelT. MicroRNA-21 regulates expression of the PTEN tumor sup-pressor gene in human hepatocellular cancer. Gastroenterology2007;133(2):647–58.

[191] Wang Y, Lee AT, Ma JZ, et al. Profiling microRNA expression inhepatocellular carcinoma reveals microRNA-224 up-regulation andapoptosis inhibitor-5 as a microRNA-224-specific target. J Biol Chem2008;283(19):13205–15.

[192] Fornari F, Gramantieri L, Ferracin M, et al. MiR-221 controlsCDKN1C/p57 and CDKN1B/p27 expression in human hepatocellularcarcinoma. Oncogene 2008;27(43):5651–61.

[193] Gramantieri L, Fornari F, Ferracin M, et al. MicroRNA-221 targetsBmf in hepatocellular carcinoma and correlates with tumor multifo-cality. Clin Cancer Res 2009;15(16):5073–81.

[194] Wong QW, Lung RW, Law PT, et al. MicroRNA-223 is commonlyrepressed in hepatocellular carcinoma and potentiates expression ofStathmin 1. Gastroenterology 2008;135(1):257–69.

[195] Li W, Xie L, He X, et al. Diagnostic and prognostic implicationsof microRNAs in human hepatocellular carcinoma. Int J Cancer2008;123(7):1616–22.

[196] Fornari F, Gramantieri L, Giovannini C, et al. MiR-122/cyclin G1interaction modulates p53 activity and affects doxorubicin sensitivityof human hepatocarcinoma cells. Cancer Res 2009;69(14):5761–7.

[197] Lin CJ, Gong HY, Tseng HC, Wang WL, Wu JL. miR-122 targets ananti-apoptotic gene, Bcl-w, in human hepatocellular carcinoma celllines. Biochem Biophys Res Commun 2008;375(3):315–20.

[198] Coulouarn C, Factor VM, Andersen JB, Durkin ME, ThorgeirssonSS. Loss of miR-122 expression in liver cancer correlates with sup-pression of the hepatic phenotype and gain of metastatic properties.Oncogene 2009;28(40):3526–36.

[199] Li N, Fu H, Tie Y, et al. miR-34a inhibits migration and invasionby down-regulation of c-Met expression in human hepatocellularcarcinoma cells. Cancer Lett 2009;275(1):44–53.

[200] Liu WH, Yeh SH, et al. MicroRNA-18a prevents estrogen receptor-alpha expression, promoting proliferation of hepatocellular carcinomacells. Gastroenterology 2009;136(2):683–93.

[201] Li S, Fu H, Wang Y, et al. MicroRNA-101 regulates expressionof the v-fos FBJ murine osteosarcoma viral oncogene homolog(FOS) oncogene in human hepatocellular carcinoma. Hepatology2009;49(4):1194–202.

[202] Xu T, Zhu Y, Xiong Y, Ge YY, Yun JP, Zhuang SM. MicroRNA-195suppresses tumorigenicity and regulates G1/S transition of humanhepatocellular carcinoma cells. Hepatology 2009;50(1):113–21.

[203] Zhang X, Liu S, Hu T, He Y, Sun S. Up-regulated microRNA-143transcribed by nuclear factor kappa B enhances hepatocarci-noma metastasis by repressing fibronectin expression. Hepatology2009;50(2):490–9.

gy/Hematology 82 (2012) 116–140 139

[204] Huang XH, Wang Q, Chen JS, et al. Bead-based microarray analysisof microRNA expression in hepatocellular carcinoma: miR-338 isdownregulated. Hepatol Res 2009;39(8):786–94.

[205] Salvi A, Sabelli C, Moncini S, et al. MicroRNA-23b mediatesurokinase and c-met downmodulation and a decreased migrationof human hepatocellular carcinoma cells. FEBS J 2009;276(11):2966–82.

[206] Kota J, Chivukula RR, O’Donnell KA, et al. Therapeutic microRNAdelivery suppresses tumorigenesis in a murine liver cancer model.Cell 2009;137(6):1005–17.

[207] Gao Y, He Y, Ding J, et al. An insertion/deletion polymorphismat miRNA-122-binding site in the interleukin-1alpha 3’ untrans-lated region confers risk for hepatocellular carcinoma. Carcinogenesis2009;30(12):2064–9.

[208] Chung GE, Yoon JH, Myung SJ, et al. High expression of microRNA-15b predicts a low risk of tumor recurrence following curativeresection of hepatocellular carcinoma. Oncol Rep 2010;23(1):113–9.

[209] Nakano S, Haratake J, Okamoto K, Takeda S. Investigation of resectedmultinodular hepatocellular carcinoma: assessment of unicentric ormulticentric genesis from histological and prognostic viewpoint. AmJ Gastroenterol 1994;89(2):189–93.

[210] Wilkens L, Bredt M, Flemming P, Klempnauer J, Heinrich KreipeH. Differentiation of multicentric origin from intra-organ metastaticspread of hepatocellular carcinomas by comparative genomichybridization. J Pathol 2000;192(1):43–51.

[211] Ng IO, Guan XY, Poon RT, Fan ST, Lee JMF. Determination of themolecular relationship between multiple tumour nodules in hepato-cellular carcinoma differentiates multicentric origin from intrahepaticmetastasis. J Pathol 2003;199(3):345–53.

[212] Potti A, Dressman HK, Bild A, et al. Genomic signatures to guide theuse of chemotherapeutics. Nat Med 2006;12(11):1294–300.

[213] Tanaka S, Arii S. Molecularly targeted therapy for hepatocellularcarcinoma. Cancer Sci 2009;100:1–8.

[214] Llovet JM, Bruix J. Molecular targeted therapies in hepatocellularcarcinoma. Hepatology 2008;48:1312–27.

[215] Zhu AX, Duda DG, Sahani DV, Jain RK. HCC and angiogenesis:possible targets and future directions. Nat Rev Clin Oncol 2011.

[216] Abou-Alfa GK, Schwartz L, Ricci S, et al. Phase II study of sorafenibin patients with advanced hepatocellular carcinoma. J Clin Oncol2006;24:4293–300.

[217] Llovet JM, Ricci S, Mazzaferro V, et al. Sorafenib in advanced hepa-tocellular carcinoma. N Engl J Med 2008;359(4):378–90.

[218] Cheng AL, Kang YK, Chen Z, et al. Efficacy and safety of sorafenibin patients in the Asia-Pacific region with advanced hepatocellularcarcinoma: a phase III randomised, double-blind, placebo-controlledtrial. Lancet Oncol 2009;10:25–34.

[219] Printz C. Clinical trials of note. Sorafenib as adjuvant treatment inthe prevention of disease recurrence in patients with hepatocellularcarcinoma (HCC) (STORM). Cancer 2009;115(20):4646.

[220] Yeganeh M, Finn RS, Saab S. Apparent remission of a solitarymetastatic pulmonary lesion in a liver transplant recipient treated withsorafenib. Am J Transplant 2009;9(12):2851–4.

[221] Saab S, McTigue M, Finn RS, Busuttil RW. Sorafenib as adjuvanttherapy for high-risk hepatocellular carcinoma in liver trans-plant recipients: feasibility and efficacy. Exp Clin Transpl 2010;4:307–13.

[222] Tanaka S, Sugimachi K, Maehara S, et al. Oncogenic signal transduc-tion and therapeutic strategy for hepatocellular carcinoma. Surgery2002;131:S142–7.

[223] Wilhelm SM, Dumas J, Adnane L, et al. Regorafenib (BAY 73-4506):A new oral multikinase inhibitor of angiogenic, stromal and oncogenicreceptor tyrosine kinases with potent preclinical antitumor activity. IntJ Cancer 2011;129(1):245–55.

[224] Tanaka S, Arii S. Current status of molecularly targeted ther-apy for hepatocellular carcinoma: basic science. Int J Clin Oncol
2010;15:235–41.

1 Oncolo

B

lSUcHHNaHhAIOndsnaas

fTrChdCPir

cIv

ChimSBaRCtot

tTOiaatsNtrYiiaMilmitih


iographies

Dr. Ashish Singhal, M.D. is a Postdoctoral Clinical Fel-ow in Division of Liver Transplantation, Department ofurgery at Temple University Hospital, Philadelphia, PA,SA. He received his medical degree at one of the best medi-

al schools in India, Himalayan Institute of Medical Sciences.e completed his surgical residency from Sir Ganga Ramospital, New Delhi, India and has earned the Diplomate ofational Board in Surgery. Further, he was associated withpremier living related liver transplant program at Apolloospital, New Delhi in capacity of Surgical Registrar. Later,e worked as a Clinical Research Fellow in the Division ofbdominal Organ Transplantation at Nazih Zuhdi Transplant

nstitute, INTEGRIS Baptist Medical Center, Oklahoma City,K, USA. His surgical interests include hepatocellular carci-oma, liver transplantation, split liver transplantation, livingonor liver transplantation, and minimally invasive liverurgery. His research interests include hepatocellular carci-oma, acute liver failure, hepatitis C, hepatic regeneration,nd transplant immunology. He has authored several clinicalnd scientific research papers and has presented his work ateveral national and international meetings.

Dr. Muralidharan Jayaraman, Ph.D. is an Assistant Pro-essor of Research at the Department of Cell Biology,he University of Oklahoma Health Science Center and a

esearcher at the Peggy and Charles Stephenson Oklahomaancer Center, The University of Oklahoma. He receivedis doctoral degree from Pondicherry University, India. Heid his postdoctoral fellowship trainings at Fels Institute forancer Research and Molecular Biology, Temple University,hiladelphia and at the Department of Cell Biology, Wash-

ngton University, St. Louis. He is currently working on theole of gep oncogenes in cancer progression.

Dr. Danny N. Dhanasekaran completed his Ph.D. in bio-
hemistry at the Indian Institute of Science in Bangalore,ndia. After completing post-doctoral fellowships at the Uni-ersity of Wisconsin at Madison and the National Jewish
ahm

gy/Hematology 82 (2012) 116–140

enter for Immunology and Respiratory Medicine in Denvere joined the faculty at Temple University School of Medicinen Philadelphia. At present, he is a faculty at the Depart-

ent of Cell Biology at The University of Oklahoma Healthciences Center as well as the Director of the Center forasic Cancer Research, Deputy Director for Basic Sciences,nd Samuel Noble Foundation Endowed Chair for Canceresearch at the Peggy and Stephenson Oklahoma Cancerenter, The University of Oklahoma Health Sciences Cen-

er, Oklahoma City, OK. His research interests are focusedn defining the aberrant signaling mechanisms that lead toumorigenesis and progression.

Dr. Vivek Kohli, M.D. is the Surgical Director ofhe abdominal organ transplant division at Nazih Zuhdiransplant Institute at INTEGRIS Baptist Medical Center,klahoma City, Oklahoma. His field of surgical expertise

ncludes transplantation of liver, kidney and pancreas. Inddition he performs complex surgeries for liver, pancreasnd bile duct related problems including cancers related tohese organs. His medical education was at the best medicalchool in India, the All India Institute of Medical Sciences inew Delhi where he received advanced training in gastroin-

estinal and hepatobiliary surgery. He completed a surgicalesidency at Stony Brook University in Long Island, Nework. He is a certified transplant surgeon by the Amer-

can Society of Transplant Surgery. He has a fellowshipn transplant surgery from the Mayo Clinic, in Minnesota,nd later gained further experience at Duke Universityedical Center, in North Carolina. His surgical interests

nclude hepatocellular carcinoma, liver transplantation, splitiver transplantation, living donor liver transplantation, and

inimally invasive approach for kidney donor, hepatobil-ary and pancreatic surgery His research interests relateo study of regeneration of the liver, its recovery afterschemia reperfusion injury, hepatitis and pathogenesis ofepatocellular cancer in relation to hepatitis viruses. He has
uthored several clinical and scientific research papers andas presented his work at several national and internationaleetings.

Documents

Molecular and serum markers in hepatocellular carcinoma: Predictive tools for prognosis and recurrence