biomarkers for virus-induced hepatocellular carcinoma (hcc)

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Infection, Genetics and Evolution xxx (2014) xxx–xxx

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Contents lists available at ScienceDirect

Infection, Genetics and Evolution

journal homepage: www.elsevier .com/locate /meegid

Review

Biomarkers for virus-induced hepatocellular carcinoma (HCC)

http://dx.doi.org/10.1016/j.meegid.2014.06.0141567-1348/� 2014 Elsevier B.V. All rights reserved.

Abbreviations: AFP, a-fetoprotein; HCC, hepatocellular carcinoma; HBV/HCV, Hepatitis B virus/C virus; DCP, des-gamma-carboxy prothrombin; GP73, Golgi proteGlypican-3; CD10, Cluster of differentiation 10; CD36, Cluster of differentiation 36; SAGE, serial analysis of gene expression; CALLA, common acute lymphatic lantigen; LC–MS, liquid chromatography–mass spectrography; PIVKA-II, proteins induced by vitamin K absence; MMP, matrix metalloproteinase; 2D-PHAGE, 2-dimphage; MALDI-TOF, matrix-assisted laser desorption/ionization; SELDI-TOF, surface enhanced laser desorption/ionization time-of-flight; FNAB, fine needle aspirationFNAC, fine needle aspiration cytology; FAT, fatty acid translocase; NAFLD, non-alcoholic fatty liver disease; CLD, chronic liver disease; CK7, Cytokeratin 7; CK19, Cyt19; NASH, nonalcoholic steatohepatitis; SFRP, secreted frizzled-related proteins; GRIPS, glypican-related integral membrane proteoglycans; ICC, intra hepatic chcarcinoma; SCCA, squamous cell carcinoma antigen; FC-GP73, fucosylated GP73; GGT, c-glutamyl transferase; AFU, a-1-fucosidase; TGF-b1, transforming growth faHCR2, human carbonyl reductase 2 enzyme; GOLPH2, Golgi phosphoprotein 2; TSGF, tumor-specific growth factor; EGFR, epidermal growth factor receptor; EGF, egrowth factor; HGF, hepatocyte growth factor; FGF, fibroblast growth factor; TNF, tumor necrosis factor; IL-6, interleukin 6; DEN, diethylnitrosamine; STAT, signal trand activator of transcription; CXCR, N-terminal cysteines of CXC chemokines receptor; TH2, T-helper 2; VEGF, vascular endothelial growth factor; miR, miRNA;scavenger receptor class B member 1; SCARB2, scavenger receptor class B member 2; PTEN, phosphatase and tensin homolog; PDC4, pyruvate decarboxylase regRECKS, reversion-inducing cysteines-rich protein with Kazal motifs.⇑ Corresponding author. Tel.: +966 535168434; fax: +966 26952076.

E-mail addresses: [email protected], [email protected] (I. Qadri).

Please cite this article in press as: Mathew, S., et al. Biomarkers for virus-induced hepatocellular carcinoma (HCC). Infect. Genet. Evol. (2014),dx.doi.org/10.1016/j.meegid.2014.06.014

Shilu Mathew a,b,c, Ashraf Ali d, Hany Abdel-Hafiz e, Kaneez Fatima f, Mohd Suhail d,Govindaraju Archunan c, Nargis Begum a, Syed Jahangir a, Muhammad Ilyas g, Adeel G.A. Chaudhary d,Mohammad Al Qahtani b, Salem Mohamad Bazarah h, Ishtiaq Qadri d,⇑a Post Graduate Department of Biotechnology and Chemistry, Jamal Mohamed College, Tiruchirappalli 620 020, Indiab Center of Excellence in Genomic Medicine Research, King AbdulAziz University, P.O. Box 80216, Jeddah 21589, Saudi Arabiac Department of Animal Science, Bharathidasan University, Tiruchirappalli 620 024, Indiad King Fahd Medical Research Center, King AbdulAziz University, P.O. Box 80216, Jeddah 21589, Saudi Arabiae University of Colorado Denver AMC, Aurora, CO 80045, USAf IQ-Institute of Infection and Immunity, Lahore, Pakistang Post Graduate Department of Botany, Jamal Mohamed College, Tiruchirappalli 620 020, Tamil Nadu, Indiah Department of Gastroenterology, School of Medicine, King AbdulAziz University Hospital, P.O. Box 80215, Jeddah 21589, Saudi Arabia

3334353637383940414243

a r t i c l e i n f o

Article history:Received 4 April 2014Received in revised form 14 June 2014Accepted 14 June 2014Available online xxxx

Keywords:BiomarkersHepatocellular carcinomaHepatitis virusesPrognosis

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a b s t r a c t

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and is advanced bysevere viral hepatitis B or C (HBV or HCV) as well as alcoholic liver disease. Many patients with early dis-ease are asymptomatic therefore HCC is frequently diagnosed late requiring costly surgical resection ortransplantation. The available non-invasive detections systems are based on the clinical utility of alphafetoprotein (AFP) measurement, together with ultrasound and other more sensitive imaging techniques.The hallmark of liver disease and its propensity to develop into fully blown HCC is depended on severalfactors including the host genetic make-up and immune responses. While common symptoms involvediarrhea, bone pain, dyspnea, intraperitoneal bleeding, obstructive jaundice, and paraneoplastic syn-drome, the evolution of cell and immune markers is important to understand viral induced liver cancersin humans. The circulating miRNA, cell and immune based HCC biomarkers are imperative candidates tosuccessfully develop strategies to restrain liver injury. The current molecular genetics and proteomicanalysis have lead to the identification of number of key biomarkers for HCC for earlier diagnosis andmore effective treatment of HCC patients. In this review article, we provide latest updates on the bio-markers of HBV or HCV-associated HCC and their co-evolutionary relationship with liver cancer.

� 2014 Elsevier B.V. All rights reserved.

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. HCC biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

in; GPC3,eukemiaensionalbiopsy;

okeratinolangio-ctor-b1;

pidermalansducerSCARB1,ulator 4;

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2.1. Protein biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.1.1. Alpha fetoprotein (AFP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.1.2. Des-gamma-carboxy prothrombin (DCP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.1.3. Golgi protein 73 (GP73) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.1.4. Neprilysin (CD10) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.1.5. CD36 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.1.6. Cytokeratin 7 (CK7) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.1.7. Cytokeratin 19 (CK19) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.1.8. Wnt pathway proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.1.9. Glypican-3 (GPC3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.2. Enzymes and isoenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.3. Growth factors and its receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.4. Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2.4.1. Interleukin 6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.4.2. Interleukin 8 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.4.3. Interleukin 10 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

2.5. Nucleic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2.5.1. Circulating mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.5.2. Solid tumor mRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002.5.3. Micro RNA (miRNA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
3. Evolutionary relationship of proactive cytokines and inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004. Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

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1. Introduction

Each year more than 500,000 new patients are diagnosed withhepatocellular carcinoma (HCC) in the world El-Serag, 2011. HCCis one of the fastest rising cancers in the region of China, SoutheastAsia as well as Africa (Parkin et al., 2005). An overview of the basicunderstanding of epidemiology, stages and its multidisciplinarycharacter of the disease is necessary to conquer the challengesfaced in clinical research (Zoulim and Locarnini, 2012). It is thethird most deadliest and fifth most common cancer across theworld. HCC is a multi-factor, multi-step and complex processwhich occurs due to persistent infection of hepatitis B virus(HBV) and hepatitis C virus (HCV) (Kumar et al., 2003). Besides,usage of alcohol, aflatoxin B1 consumption is another etiologicalagent in HCC. The fatality rate is quite higher due to rapid tumorprogression. In countries where hepatitis is not endemic, most ofthe HCC are not primary but metastasis of cancer from other partsof the body. Hepatitis is the inflammation of the liver caused byhepatitis virus. It can also be caused by other infections, toxic sub-stances and autoimmune diseases. There are 6 different types ofhepatitis viruses including A, B, C, D, E, and G, from which typeA, B, and C are the most common. Hepatitis B is transmittedthrough blood and infected body fluid while hepatitis C virus isspread only through exposure to an infected person’s blood(Mizejewski, 2001). Hepatic resection or transplantation is the onlycurative treatment available now for HCC patients (Mizejewski,2001). Due to its asymptomatic nature, early detection of HCC isdifficult and many patients present with advanced form of the dis-ease at diagnosis and the prognosis for such patients remain poor.This review summarizes recent studies of potential biomarkers fordiagnosis and to monitoring metastasis or recurrence of HCC.

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2. HCC biomarkers

Biomarkers detectable in blood, urine, or tissue samples are usedas molecular indicators for various types of diseases and their man-agement. Their thresholds concentrations are used to detect thepresence of various cofactors responsible for activating the diseases.Fluctuations in threshold concentrations have resulted in the pro-spective disease progression, diagnosis and guide therapy. Severalbiomarkers have been recognized for different diseases as well as

cite this article in press as: Mathew, S., et al. Biomarkers for virus-indi.org/10.1016/j.meegid.2014.06.014

research studies are still ongoing to fully understand and evaluatethe clinical significance of utilizing such biomarkers. A lot of moneyand time can be saved as compared to empiric or other broad treat-ment approaches. Biomarkers could be more useful as a measure-ment tool to detect presence and progression of diseases,ultimately guiding them towards more targeted therapy. Under-standing these markers is quite successful in detecting several typeof cancer. They can also be used effectively in case of HCC. Molecularbiomarkers were discovered using scientific platforms such asgenomics along with proteomics. Fig. 1 depicts the various path-ways activated when HCC is induced. Apart from genomics and pro-teomics platforms, biomarker assay techniques such as glycomics,metabolomics, secretomics and lipidomics are the most commonlyused as techniques in identification of biomarkers. Northern blot,Gene expression, SAGE and DNA Microarray, Proteomic Approachinvolves 2D-PAGE, LC–MS, SELDI-TOF (or MALDI-TOF), Ab Micro-array and Tissue Microarray are involved to develop the screeningof biomarkers at various grades of the disease.

2.1. Protein biomarkers

2.1.1. Alpha fetoprotein (AFP)For decades, the most commonly used biochemical blood test to

detect liver cancer is by screening for alpha fetoprotein (AFP). AFPis a glycoprotein with a molecular weight of 70 kDa, secreted byimmature fetal liver cells and appears in cancer cells. AFP acts asa transporter molecule for several ligands, such as fatty acids, phy-toestrogen, heavy metals, retinoid, steroids, flavonoids, dyes, bili-rubin, dioxin and various drugs (Mizejewski, 2001). AFP isthought to exhibit immunosuppressive activity; as it plays a vitalrole in regulation of cell proliferation (O’Neill et al., 1982). Thisplasma protein is synthesized by yolk sac and by the liver. It isone of the chief proteins secreted during the fetus developmentand is present in very low levels in adults. Therefore AFP is oneof the several tumor markers that are present at high level whena person is affected by cancer. It is mainly found in nonseminoma-tous germ cell tumor and in liver cancer. Patients with cirrhosis orchronic hepatitis also are reported with higher blood levels ofalpha-feto protein (Fattovich et al., 2004). The level of AFP is higherin early stages of HCC, but it normalizes as disease progresses(Llovet et al., 2008). In general, it has been indicated serum AFPlevel more than 500 ng/mL, indicating the presence of HCC but

uced hepatocellular carcinoma (HCC). Infect. Genet. Evol. (2014), http://



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Fig. 1. Schematic diagram depicting activation of various pathways activated and various signature molecules induced from HCC.

Fig. 2. Phylogenetic tree shows the evolutionary relationship of AFP among different species. Branch length is indicated in percentage (colored red). This tree was generatedby using Multiple alignments: MUSCLE, alignment curation: Gblocks, construction of phylogenetic tree: PhyML and visualized by TreeDyn. Phylogram was generated by usingapproximate Likelihood-ration Test (aLRT): SH-like. The number below the Phylogram denotes the unit change in the amino acid sequence among the indicated species. (Forinterpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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lower AFP are also seen in benign hepatocellular carcinoma(Mizejewski, 2001). AFP is further subdivided into three differentforms of glycoforms such as AFP-L1, AFP-L2 and AFP-L3, in whichAFP-L3 is associated with large mass of cancer tissue, poor differ-entiation and malignant features (Lin et al., 2004). AFP-L3 is mostlyuseful in the differential diagnosis of individuals with total serumAFP 6 200 ng/mL, which may result from a variety of benignpathologies including chronic liver diseases (Zabora et al., 2001).

A close evolutionary relationship was studied between some ofthe species synthesizing AFP. The FASTA sequence of AFP wasretrieved from UniProtKB for different species and the phyloge-netic analysis was done by using Phylogeny.fr (Anisimova andGascuel, 2006; Dereeper et al., 2008, 2010; Guindon and Gascuel,2003; Castresana, 2000). Fig. 2 shows an evolutionary relationshipof AFP among different species with their Uniprot ID. Human,chimpanzee and gorilla are closely related as the branch lengthsleads to the node. Rat and mouse protein sequence appears to be

Please cite this article in press as: Mathew, S., et al. Biomarkers for virus-inddx.doi.org/10.1016/j.meegid.2014.06.014

a close relative of AFP, whereas the branch length for chicksequence is so long may be due to high amount of AFP sequencedivergence.

2.1.2. Des-gamma-carboxy prothrombin (DCP)DCP is an abnormal prothrombin protein induced by antagonist

II (PIVKA-II), discovered in 1984 and its level increases in case ofliver cancer (Liebman et al., 1984). DCP represents an abnormalproduct of liver carboxylation during the formation of thrombogenthat acts as an autologous mitogen for HCC cell lines (Ikoma et al.,2002; Suzuki et al., 2005). Previous studies have shown that thecombination of serum AFP and DCP was better for detecting HCCthan using either AFP or DCP alone (Zhu et al., 2013).

2.1.3. Golgi protein 73 (GP73)GP73 is a 73 kDa transmembrane glycoprotein that normally

resides within the golgi complex. It is expressed in normal biliary




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epithelial cells whereas normal hepatocytes do not express thisprotein, and its expression is significantly increases in liver dis-eases including HCC (Kladney et al., 2002). Serum GP73 is a valu-able biomarker for patients with HCC (Riener et al., 2009; Maoet al., 2010).

2.1.4. Neprilysin (CD10)Neprilysin (CD10) is metalloprotease enzyme present on cell

surface, composed of 90–110 kDa and located at 3q21–27 of thehuman chromosome (McIntosh et al., 1999). It is enrolled in inac-tivating bioactive peptides. CD10 are also called as common acutelymphatic leukemia antigen (CALLA) plays a significant role indegrading the abnormal misfolding of amyloid beta sheets in nervetissue (McIntosh et al., 1999). CD10 is considered in differentiatingB cells, T cells as well as natural killer cells (Galy et al., 1995). CD10antigen has been reported on the surface of normal early lymphoidprogenitor cells, germinal center B cells within lymphoid tissueand in immature B cells (McIntosh et al., 1999). Abnormal methyl-ation of CD10 results in expression of surface proteins in normalcells, precursor B cells and neoplastic B cells (McIntosh et al.,1999). Therefore loss in CD10 will increase the migration, growthand survival of cells contributing to neoplastic development andprogression (Papandreou and Nanus, 2010). It is considered as animportant marker for expression in B cell for hepatocellular andrenal cell carcinoma. The expression of CD10 is more in metastaticmelanomas rather than primary carcinoma (Bilalovic et al, 2004).The persistence of heavier inflammation in the liver is one of thecommon problems faced in HCC (Jia et al., 2007). Many genesencode for cytokines which are produced by immune cells to com-municate either up or down regulation of the immune response(Jia et al., 2007). Normal liver tissue had a specific pattern inimmune cell activities as they are involved in increasing the cyto-kines responsible for anti-inflammatory response and suppressingthe immune action (Jia et al., 2007). Mainly the invasive role ofcancer levels is necessary by stromal cells, which has both stimu-latory and inhibitory factors that can associate functions like cellu-lar adhesion, cell migration and expression of genes (Bemis andSchedin, 2000; Sternlicht et al., 1999; Tran et al., 1999), similarlythe identical structure of CD10 with matrix metalloproteinase’s(MMPs) can facilitate a root for tumor cell growth as well as metas-tasis (Basset et al., 1994; Talvensaari-Mattila et al., 1998). Around55 cases from United States were diagnosed with HCC and werestudied further from formalin fixed sample; paraffin embeddedblocks obtained by FNAB (fine needle aspiration biopsy) from theliver, and was immunostained using monoclonal antibody againstCD10 antigen, out of which 86% (22 HCC patients) showed positivefor canalicular staining pattern for CD10 whereas 13% (23 meta-static cancers) were positive for cytoplasmic and membranousstaining pattern (Lin et al., 2004). Study comprising from liverbiopsies of 164 UK patients, the CD10 stain canalicular pattern

Fig. 3. Phylogram analysis for CD10 (colored purple) and CD36 (colored blue) species. Thamong the indicated species. (For interpretation of the references to color in this figure


was reported in liver tissues with mild fibrosis and inflammation,which becomes drastically down with increase in fibrosis or lobu-lar inflammation (Shousha et al., 2004). Comparing samplesextracted by FNAC (fine needle aspiration cytology) of 22 HCCcases (7 cases of HCC and 15 cases of metastatic) from India, alsoreported 68% positive for CD10 canalicular staining pattern(Ahuja et al., 2008). CD10 expressions is also clearly seen in differ-entiating HCC and non HCC cases by immunostaining antibodiesagainst CD10 for 63 HCC cases and 25 non-HCC cases from Ger-many (Borscheri et al., 2001). Therefore CD10 is a useful biomarkerstain for specifically determining HCC versus non HCC warrantsfurther study (Chu and Arber, 2000).

2.1.5. CD36CD36 known as fatty acid translocase (FAT) is a membrane

protein, present as fourth glycoprotein on the platelet surface asthey are grouped under proteins that involve in lysosomal integralmembrane protein II and SR-BI (type B scavenger receptors)(Abumrad et al., 1993; Janssen et al., 2001). Being a multifunctionalcomplex, it takes part in lipid transport, removes apoptotic cells, celladhesion, controls inflammation, atherosclerosis as well as diabetes(Silverstein and Febbraio, 2000). One of the major activities of CD36is experimentally known for lipid homeostasis (Febbraio et al.,2002). Recent studies have noticed CD36 linked to synthesis of pros-taglandin E2, calcium flux ion channels and phospholipase activa-tion (Kuda et al., 2011). In tumor tissue, the expression of CD36 ispoised peculiarly in distinguishing multiple cell type phenotypessuch as promoting production of foam cells, activating cytokines,reactive oxygen species and blocks migration (Silverstein andFebbraio, 2009). This reduced expression of CD36 even in dendriticcells, also mediates extracting apoptotic cells thereby allowingtumor antigens into cytotoxic T cells, which results in attackingthe immune system (McDonnell et al., 2010). Therefore any modu-lation in CD36 expression will actively spread carcinoma. CD36 ishighly expressed in endothelial cells but spotting of this moleculewas done using a novel antibody named MO30, which reacted withmicrovilli of cultured hepatoma and melanoma cells (Maeno et al.,1994). The expression of CD36 in 66 cases affected with HCV and32 non diseased liver cases from Spain showed CD36 up regulatedand linked with increase steatosis, hyperinsulineamia and insulinresistance (Pez et al., 2013). It has been found that translocation ofCD36 fatty acid to the plasma membrane results in depositing liverfat in HCV and non-alcoholic fatty liver disease (NAFLD) affectedpatients (Miquilena-Colina et al., 2010). It has been recently sug-gested that the serum CD36 levels reflect the severity of CD36expression on the Kupffer cells in patients with HCV-related chronicliver disease (CLD-C), and that the serum CD36 levels were associ-ated with obesity (Himoto et al., 2013).

Evolutionary study for different species synthesizing CD10 andCD36 is depicted in Fig. 3. The presence of overlapping species

e number below the Phylogram denotes the unit change in the amino acid sequencelegend, the reader is referred to the web version of this article.)




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names for CD36 represents its sequence closely related branchlengths leading to nodes are very close to zero. Similar is notedfor CD10, except for C-elegans that do not appear to be closestrelative.

2.1.6. Cytokeratin 7 (CK7)Cytokeratin are proteins consisting of intermediate filament pro-

teins, also known as keratins (Schweizer et al., 2006). They areexpressed in both pairs of keratinized and non-keratinized epithelialcells, which constitute about 85% of mature keratinocytes. Thereactive role is to take part in differentiation, tissue specializationand managing the structural function within the epithelial cells.Cytokeratin 7 (CK7) is also called as sacolectin, which is categorizedunder the subgroup of glandular epithelia and its tumors, similarlywith transitional epithelium and transitional carcinoma(Monoclonal Antibodies to Cytokeratins). Cytokeratin also plays akey role by interfering production of interferon-dependent second-ary proteins thereby reversing interferon induction and storing cells.It is present on the membrane surface of both normal andtransformed cells (Cytokeratin 7 Antibody (LP1K)). The study ofintermediate expression of filaments, for the cytokeratin in fibrola-mellar carcinoma was done by using paraffin sections, reacted withmonoclonal antibodies to provide signals, that can communicatewith epitopes shared by cytokeratin polypeptides (Van Eykenet al., 1990). A case study was presented from Germany for twopatients affected with HCC, were CK7 was strongly expressed infibrolamellar carcinoma of the liver, where as in normal tissue it ispresent only in bile ducts (Van Eyken et al., 1990). Western blottingstudies of the lymph node metastasis further confirmed the immu-nohistochemical data for CK7 (Van Eyken et al., 1990). An immuno-histochemistry study done for 20 HCC cases from USA, in whichepithelial cell adhesion cells showed positive expression for boththe CK7 and CK9 for about 53% and 26% (Krings et al., 2013). Matsu-ura et al. have reported the higher level of immunohistochemicalexpression of CK7 in scirrhous HCC than in ordinary HCC(Matsuura et al., 2005). Similarly Fanni et al. have used CK7 to distin-guish HCC from peripheral cholangiocarcinoma (CC) Fanni et al.,2009, suggesting that CK can be used as biomarkers for certain cat-egories of epithelial differentiation of cells (Van Eyken et al., 1990).

2.1.7. Cytokeratin 19 (CK19)Cytokeratin 19 (CK19) is a 40kda protein and involved in struc-

tural integrity by maintaining the filament proteins. It is locatedspecifically at the periderm, a layer that coats the epidermis.Keratin 19 is involved in signal transduction of B-lymphocyte acti-vation, development and differentiation (Chu and Weiss, 2002).Therefore CK19 is a chronic keratins expressed in carcinoma,cleaved by caspase3 that releases the soluble fragments whichare detected in cancer patients (Pujol et al., 1993). Antibodies toCK19 were diagnosed for 32 HCC cases from Hong Kong, wereanti-CK19 produced distinctive staining of the bile ducts (Leonget al., 1998). CK19 was reported to be expressed in HCC and is cor-related with HCC metastasis and recurrence. It has also been

Fig. 4. Phylogenetic tree depicting relationship between CK7 and CK19 (colored green areader is referred to the web version of this article.)


reported as a prognostic marker in HCC after operation (Yanget al., 2008).

Evolutionary relationship between CK7 and CK19 secreting spe-cies is indicated in Fig. 4. There exist a close relation betweenmouse and rat with CK19, showing closeness towards humanCK19 sequence, where as for CK7 represents strongly relatedsequence between rat and mouse protein sequence as well asamong human and chimpanzee FASTA sequence as their branchlength leading to these nodes are very close. The unit change inamino acid sequence among the species is 0.4%.

2.1.8. Wnt pathway proteinsWnt signal transduction pathway is made up of proteins that

pass signals from outside of a cell through cell surface receptorsto inside of the cell (Cadigan and Nusse, 1997). These proteinsare secreted by lipid-modified glycoproteins that are 350–400amino acids in length. Wnt signaling was first recognized for itsrole in carcinogenesis, but has since been known for its functionin embryonic development. Activation of this pathway results intwo different cascades called non-canonical and canonical andthereby involving beta-catenin proteins (Pez et al., 2013). Deregu-lation is an earlier indication in HCC resulting in major aggressivephenotype. Transcriptomic and metabolomic datasets from humanliver tissue representing nonalcoholic fatty liver disease (NAFLD)progression from normal, steatosis, nonalcoholic steatohepatitis(NASH) and compared to published data for HCC from USA. Thesedata indicate an overlap in the pathogenesis of NAFLD and HCCwhere several classes of HCC related genes and metabolites arealtered in NAFLD. Wnt signaling and several metabolites are differ-ent, thus implicating these genes and metabolites as mediators inthe transition from NASH to HCC (Clarke et al., 2014). Anothergenotyping study from USA consisting of 425 chronically infectedHCV veterans reported the activation of several genes from Wntsignaling pathway thereby resulting in hepatic fibrosis (Liu et al.,2013). An epigenetic study from China from secreted frizzled-related proteins (SFRPs), one of the antagonists for Wnt signalingpathway, was found to be down regulated in hepatocarcinogenesis.Their study also suggests the silencing of SFRP1 constitutes activa-tion of Wnt signaling pathway (Quan et al., 2013). Whole genomesequencing of 88 tumor/normal cases from USA has determined62.5% of Wnt pathway altered resulting as one of the major onco-genic drivers for HCC (Kan et al., 2013). Therefore, the suppressionof Wnt/beta-catenin signaling pathway exerts pressure on rapidproliferation and apoptosis of liver tissues.

2.1.9. Glypican-3 (GPC3)Glypican 3 (GPC3) is member of protein family coded by GPC3

gene (Filmus and Capurro, 2013). These glypican-related integralmembrane proteoglycans (GRIPS) are connected to the surface ofcell through glycosyl-phosphatidylinositol linkage. They play amajor role is to regulate several growth factors as well as it hasthe capacity to stimulate or inhibit its action through signaling

nd red box). (For interpretation of the references to color in this figure legend, the




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receptors (Glypican-3). GPC3 is expressed commonly in fetal livercells and placenta but not expressed in adult liver tissue(Shafizadeh et al., 2008). Several studies have shown that the levelsof GPC3 were higher in the serum of HCC patients in comparisonwith benign liver disease and healthy donors, suggesting GPC3 asa possible tumor marker for HCC (Febbraio et al., 2002; Anatelliet al., 2008). GPC3 is involved in stimulating tumor growth by acti-vating the Wnt signaling pathway (Gao and Ho, 2011). Patientswith hepatitis have higher percentage of GPC3 positive comparedwith GPC3 negative (Yu et al., 2013). Immunohistochemistry stain-ing for 80 resection HCC cases from USA, also identified that theGPC3 was sensitive for diagnosing HCC but its poorly seen in highlywell differentiated hepatocytes and fibrolamellar variant of HCC(Shafizadeh et al., 2008). In China, study was done by using 7D11mAb for GPC3 in normal as well intra hepatic cholangiocarcinoma(ICC) samples, revealing 85% of expression of Glypican suggestinglarger scale of clinical diagnosis can be done (Yu et al., 2013).

2.2. Enzymes and isoenzymes

Isoenzymes play an important role in tuning metabolism fordeveloping tissue in the body. As we discussed earlier DCP levelhas better correlation with tumor resection and is known to be bet-ter marker for tumor (Weitz and Liebman, 1993). In the compara-tive study of various HCC markers, DCP is found as the most usefulin predicting HCC with less sensitive to risk factors, like liver cir-rhosis and thereby differentiates HCC from non-malignant carci-noma (Volk et al., 2007). Similarly, c-glutamyl transferase (GGT)in healthy cells are synthesized from bile duct, hepatic kupfferbut it is more active in HCC tissue (Cui et al., 2003). They are con-centrated in the liver to metabolize toxins and other drugs but arealso present in gall bladder, kidney, spleen and pancreas. GGT testsmostly involve differentiating enzyme level in bile duct or liver dis-order. A lysosomal enzyme called serum a-1-fucosidase (AFU) isinvolved in hydrolyzes fucose glycosidic linkage of glycol lipidsand proteins in normal cells. It is reported to be elevated in serumof HCC patients. Measurement of AFU is beneficial in early diagno-sis of HCC along with AFP (Tangkijvanich et al., 1999; Wei et al.,2000). Human carbonyl reductase 2 enzyme (HCR2) also called asDCXR is a detoxification enzyme against alpha-dicarbonyl andreactive oxidative stress in HCC and identified to be inversely cor-related to different grades in HCC affected patients (Liu et al.,2006). It has been reported that disturbance in HCR-2 relateddetoxification is an important pathway towards progression ofHCC in Chinese patients measured by immunohistochemistry andwestern blot techniques (Liu et al., 2006). Finally, Golgi phospho-protein 2 (GOLPH2) is type II transmembrane protein present atcis and golgi cisternae, with its expression in human is witnessedin epithelial cells but in a poorly defined conditions, as it is cleavedand transported to extracellular spaces (Kim et al., 2012). This pro-tein is detected with high sensitivity compared to AFP in the serumof HCC patients (Kim et al., 2012).

2.3. Growth factors and its receptors

Growth factors are naturally occurring substances that stimu-lates cellular growth, cellular differentiation and proliferation(Growth Factor). Different types of growth factors and theirbioactive roles are discussed in this section. Transforming GrowthFactor-Beta (TGF-b) is involved in polypeptide signaling moleculeand are predominant form of growth factor present in humans.TGF-b plays an important role in the development of HCC. It isreported that TGF-b levels increased in cirrhotic livers of HCCpatients (Okumoto et al., 2004). Tumor-specific growth factor(TSGF) is released by malignant tumor in the serum during the pro-liferation of the cells. Therefore, the serum levels of TSGF can be used


as a marker for the presence of cancerous cell with high accuracy(Zhou et al., 2006). The epidermal growth factor receptor (EGFR) istransmembrane tyrosine kinase with its receptor family whichincludes erbB-1, c-erb-2, c-erb-3 and c-erb-4. These receptors bindto EGF, heparin-binding EGF and TGF-alpha family and associatedwith early recurrence in HCC (Ito et al., 2001). EGFR system acts as‘signaling hub’ where diverse types of survival signals and epidermalgrowth signals converge (Berasain et al., 2011). Hepatocyte growthfactor (HGF) is a cytokine that facilitates multiple from embryonicdevelopment till liver regeneration. It is produced by various tissuessuch as neoplasms and it provides stimulus for movement of malig-nant cells by autocrine and paracrine mechanism. The HGF receptoris identified as oncogene c-met key to facilitate cell invasion. It is rec-ognized as imperative not only for liver tissue growth but includesmetastasis also (Jiang et al., 1993). Therefore HGF involved in molec-ular activity in hepatic carcinoma and reduced overall survival rate(Osada et al., 2008). Fibroblast growth factor (FGF) is a soluble hep-arin-binding polypeptide, whose elevated levels are also known fordecreased disease free-survival (Poon et al., 2001). FGF is reported toincrease the synthesis of plasminogen activators and collagenases incultured cells, which resembles tumor invasion (Montesano et al.,1986). Since these FGF have strong roles in cell migration and prolif-eration, its expression have vital character in the development ofsolid cancerous tumor, where a sustained vascular platform is essen-tial (Chow et al., 1998). Targeted inhibition of fibroblast growth fac-tor (FGF) with lenalidomide showed promising activity in HCC cases(Safran et al., 2013). Recent study has reported that FGF proteinexpression is an effective predictor of early recurrence and a markerfor poor prognosis of HCC (Hyeon et al., 2013).

2.4. Cytokines

Cytokines are small, 5–20 kDa, proteins that has an importantrole in cell signaling. Cytokines involve as interferons, lympho-kines, interleukins, tumor necrosis factor (TNF) and chemokines.They are synthesized by wide range of cells including mast cells,macrophages, endothelial cells, stromal cells, B lymphocytes andT lymphocytes as well as fibroblast cells (Lackie, 2010). These sig-naling molecules mediated by receptors that act as a pillar forimmune system to spread responses to the infected part of cells.Liver is known as the central region of cytokine due to hepatocytesthat are highly susceptible to cytokine activity in various patho-physiological and physiological processes. However, the non-parenchymal kupffer cells (KCs) are able to produce cytokines thatcan act on other parts of the body (Ramadori and Armbrust, 2001).There is high evidence that cytokines inflammate and causes apop-tosis as well as necrosis of liver cells. On the other lane, they are asexperts for liver tissue regeneration after injury (Ramadori andArmbrust, 2001). Therefore, inhibition of this mediator mightimpair hepatic recovery system.

2.4.1. Interleukin 6Interleukin (IL-6) is a multifunctional cytokine that plays a crit-

ical role in hematopoiesis, as well as in the differentiation andgrowth of different cell types such as endothelial cells, neuronalcells, keratinocytes, ostoclasts and ostoblasts. They are secretedby T cells, macrophages to stabilize immune response. Smoothmuscle cells also produce cytokines for pro-inflammatory actions.IL-6 blood serum level has been reported elevated in patients withsevere liver diseases such as alcoholic hepatitis, viral hepatitis(HCV and HBV) as well as steatohepatitis. Wong et al. has shownthat patients who subsequently developed HCC had raised IL-6 lev-els 2–3 years before HCC development (Wong et al., 2009). Disrup-tion of IL-6 gene in transgenic resulted in reduction DEN-inducedhepatocarcinogenesis suggesting that IL-6 play an significant rolein HCC (Naugler et al., 2007). A study done by Cressman et al.,




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Fig. 5. Evolutionary tree of interleukin family members. The unit change in the amino acid sequence among the IL species is 1%. Each group of IL8, IL6 and IL10 are coloredblue, brown and green box. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)



27 June 2014

reports targeted disruption of IL-6 done for mice, that impairedliver regeneration, followed by liver necrosis and failure. IL-6 defi-cient mice returned with STAT3 binding, gene expression hepato-cyte proliferation when treated again with IL-6 and thusprevented liver damage. As a result, ensuring IL-6 as a critical com-ponent for liver regeneration (Cressman et al., 1996).

2.4.2. Interleukin 8Interleukin 8 is a pro-inflammatory cytokine involved in the cel-

lular response to inflammation. IL-8 is produced by a wide variety ofcells, including neutrophils, monocytes, fibroblasts, and endothelialcells (Wang et al., 2014; Yahya et al., 2013). IL-8 mediates its biolog-ical function by interacting with specific G-protein-coupled recep-tors such as CXCR1 and CXCR2. A study explored in Chinesepopulation for IL8 polymorphism with HCC cases, resulted in highserum level compared to healthy controls (Welling et al., 2012).Recent Egyptian study on patients with chronic Hepatitis C virus(HCV) infection has shown that serum levels of IL-8 were higher inpatients with HCV-associated HCC compared with control subjects(Elewa et al., 2010). Similarly, high levels of IL-8 have been foundin study performed on 90 HCC cases and 180 cirrhotic controls fromUSA (Welling et al., 2012). Therefore, the ability to measure IL-8 levelin serum could be a useful marker of HCC in patients.

2.4.3. Interleukin 10Interleukin 10 (IL-10) is a pleiotropic cytokine produced by T-

helper 2(TH2), macrophages and B-lymphocytes and can stimulateor inhibit the immune response (Yin et al., 2011). Recently, it hasbeen reported that the serum levels of IL-10 is significantly ele-vated in patients with HCV-associated HCC and IL-10 take part inthe development of HCC with suppression of immune response.These immune suppressive effects of IL-10 may play a critical rolein the development of HCC by suppressing interferon productionand enhancement of tumor cells metastatic potential. It has beenshown that IL-10 values are correlated significantly with the tumorsize suggesting that IL-10 levels can be used as tumor markers andcontribute to the deferential diagnosis in HCC patients (Othmanet al., 2013).

Evolutionary relationship for IL-8, IL-6 and IL-10 was also ana-lyzed by retrieving protein sequence from Uniprot (See Fig. 5).Clades of IL-10, IL-6 and IL-8 are nested within one another, forming


a nested IL hierarchy. The lengths of node represent the substitutionrate defined in percentage of substitution for alignment length.Branch length is indicated in percentage (colored red). The branchlength for human IL-8 sequence shows closer identity towards mon-key, rhesus monkey and macaque monkey compared to other spe-cies which are diverged high. IL-6 also depicts closer variationwith human IL6; whereas IL-10 has wide distribution of amino acidsequence between chick, human, mouse as well as rat sequence.

2.5. Nucleic acids

2.5.1. Circulating mRNACirculating mRNAs provide a useful marker to investigate vari-

ous pathological and physiological conditions of HCC patients andcan be used for early diagnosis. Using reverse-transcription poly-merase chain reaction (RT-PCR), AFP mRNA has been detected inblood circulation in HCC patients (Matsumura et al., 1994). Hepa-titis B infected patients were strongly associated with AFP mRNAin blood (Montaser et al., 2007). Blood circulating Insulin-LikeGrowth Factors I and II (IGF-II) mRNA had diagnostic value forHCC, as they are necessary for development and liver regulation.Their expression level was studied in rats during hepatic develop-ment and carcinogen, with high levels of IGF-I mRNA detectedcompared to IGFII mRNA (Himoto et al., 2005). c-Glutamyl trans-ferase (GGT) mRNA is present in liver tissues and serum of healthyhumans as well as in patients with different grades of liver disease.They are further classified into type A and type B, in which type BGGT mRNA are spotted in cancerous and non cancerous tissuesample but with poor outcome (Behne and Copur, 2012). AlbuminmRNA is another type synthesized by liver to detect in plasma andis known to be a sensitive diagnostic marker for HCC. It can bedetected by using in-situ hybridization in paraffin embed tissueand shows positive for non-cirrhotic, focal nodular hyperplasia,hepatocellular carcinoma, hepatoblastoma and hepatocellular ade-nomas (Behne and Copur, 2012).

2.5.2. Solid tumor mRNAIt has been shown that RNA expression profiles are useful mark-

ers for classification of solid tumors including HCC. Several inves-tigators have reported that livers from HCC patients have adifferent expression profiles in comparison with the livers from




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control subjects Kim et al. reported a molecular signature contain-ing 273 significantly altered genes in HCC. Among them, 12 genesencoded secretory proteins detectable in sera, which may be usedas markers for early diagnosis of HCC (Jia et al., 2007; Kim et al.,2004; Budhu et al., 2006).

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2.5.3. Micro RNA (miRNA)MicroRNAs (miRNAs) are small noncoding RNAs involved in the

regulation of gene expression (Gramantieri et al., 2008). They aresynthesized from their respective genes or from introns. Theirfunction is activated by base pairing with mRNA complementarysequence or mRNA degradation thereby resulting in gene silencing(Matsumura et al., 1994). About 1000 miRNAs are encoded in ourhuman genome (Montaser et al., 2007). These molecules act asoncogenes or tumor suppressor genes by controlling the key pro-teins associated with cell signaling pathway in cancer metabolism(Himoto et al., 2005). Recently, it has been reported that miRNAmight be very useful biomarkers, because they are very stableand are present in the blood (Giordano and Columbano, 2013). Pre-clinical and clinical studies demonstrated that cancer affects thelevels of circulating miRNA and that specific miRNAs can be asso-ciated with specific tumors (Giordano and Columbano, 2013). InHCC, miRNAs have been shown to be involved in the deregulationof many signaling pathways including p53, P13K/AK/mTOR, WNT/b-catenin, MET, MYC, RAS/MAPK and transforming growth factors(Bartel, 2009). Recent miRNA expression profiles have identifiedmolecular signatures associated with progression, prognosis, andresponse to treatment. Recent study has found three up regulatedmiRNAs in HCC samples, whereas five were down regulated. Inaddition, the expression levels of miR-92, miR-20, and miR-18were inversely correlated with the degree of HCC (Murakamiet al., 2006). Several miRNAs have been reported to be involvedin HCC including miR-142, miR-223, mi-122, miR-199, miR-221and miR-21 (Chen and Rajewsky, 2007). The expression level ofmiR-21 is highly elevated in human cancers (Yu et al., 2013;Bartel, 2009) and thereby promoting carcinoma by targeting PTEN,PDCD4 and RECKS (miRBase, 1824). Similarly high level of expres-sion is all viewed in culture supernatants of HCC cell lines withmiR-15b, miR-21, miR-130b and miR-183 (Morozova et al.,2012). miRNA is not only found in serum and plasma but also theyare found in the urine of HCC patients suggesting that more workhas to be done to select the best type of samples to be used fordiagnosis (Callegari et al., 2013).

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3. Evolutionary relationship of proactive cytokines andinflammation

Understanding the relationship between immunology, diagno-sis and treatment for viral disease is a critical factor. Large animalsare interesting group of studies, as they represent great diversity inthe control of innate immunity and its dependence on adaptiveimmunity (Bubanovic and Najman, 2004). Cytokines are key mole-cules responsible for host response to various types of infections,immune response, inflammation as well as trauma (Dinarello,2000). These molecules make disease worse (proinflammatory)as well as serve in rescuing inflammation and promote healing(anti-inflammatory) Dinarello, 2000. They have been identifiedand extensively studied in mammal, but a very few knowledge isknown about their presence in vertebrate group (Bubanovic andNajman, 2004). Homolog’s of cytokine has also been found withinjawless vertebrates (Bird et al., 2002). Cytokines such as IFN, IL andTGF-b is identified in reptiles, birds, amphibians as well as bonefish (Bubanovic and Najman, 2004). IL-10 related T cell-derivedinducible factor (IL-TIF) was recognized to have 79% amino acidhomology to humans by two groups and designated as IL-22


(Kumar et al., 2013). Wide evidence has suggested close functionalrelationship and a common evolutionary origin for alpha-feto pro-tein and albumin as they have similar gene structure (Dugaiczykand Harper, 1983). Baker et.al compared rat, mouse, bovine andhuman AFP domain with albumin domain, by computer programdesigned to quantify relationship origin of the protein, revealingthat each domain is much conserved. This study suggested thechange in amino acid substitution among the domains of albuminand AFP during the past 400 million years, since they divergedfrom common ancestors (Baker, 1988).

A comparative study for CD36 protein structure and gene loca-tions were determined by using data from various vertebrate gen-ome projects, that shared about 53–100% uniqueness as comparedto 32% identity with CD36 super family members like SCARB1 andSCARB2 (Holmes, 2012). A phylogenetic analyses was examined tostudy the potential relationship and origins of CD36 gene espe-cially SCARB1 and SCARB2 in vertebrates, which propose thatCD36 originated in ancestral genome and further duplicated subse-quently resulting in three vertebrate CD36 gene family memberssuch as CD36, SCARB1 and SCARB2 (Holmes, 2012). Qadri et al.have shown activated expression CD3 in HCV expressing cellsand this protein may be exploited in future to explore as possiblemarker in HCV-induced liver disease (Qadri et al., 2012).

With the evolution of vertebrates, there seems dramaticallygrowth of regulatory cell with its multiple functions in controllingof immune response. It has been studied in lower vertebrates, cyto-kine network of immune response consist cytokines which aremainly present in mammal known to be pro-inflammatory cyto-kines (Dinarello, 2000). Evolution relationship of cytokines ofimmune system in birds is mostly similar to mammalian(Bubanovic and Najman, 2004). Diversification of cytokines withthe evolution of suppressive cytokines, and their control mecha-nism can be defined as another output of adoptive immunity(Dinarello, 2000). There are important correlations between adop-tive immunity as well as multiple cytokines in various classes ofvertebrates, which associate anti-tumor immunity failure and fore-most source for different incidence of neoplasm in vertebrates.

4. Summary and conclusions

Treatment and management of HCC continues to be a challeng-ing task. There are many molecular abnormalities involved in thedevelopment and progression of HCC. Current molecular researchconfirmed that HCC tissue from different individuals have variousphenotypic differences. However the features that unite HCC alongwith HBV and HCV infections may result in improved hepatocyteturnover that occurs to replace immunologically affected cells(Block et al., 2003). Primary hepatocellular carcinoma (PHCC)refers to HCC originally within the organ liver (Block et al., 2003).There occurs high mortality associated with HCC due to non-cap-sular region of liver is deficient from sensory fibers and thereforeresults in symptoms for HCC repeatedly occur late PHCC, resultingin 5-year survival rate of less than 5% (Mason and El-Serag, 1999).Individuals persistently infected with HBV have a risk of death toPHCC between 10% and 25% (Mason and El-Serag, 1999; Evanset al., 1998; Montalto et al., 2002). Lifetime risk patients chroni-cally affected with HCV are between 2% and 7% (Bisceglie, 1997;Liang et al., 2000).

To date, many studies have investigated the oncogenic pathwaymechanism behind development of HCC thought to accelerate thechance has been identified are Wnt/beta catenin, c-met, phosphoin-ositol-3kinase, myc and cellular proliferation (El-Serag andRudolph, 2007). Activation of AKT signaling promotes tumor forma-tion by suppressing transforming growth factor induced apoptosis(El-Serag and Rudolph, 2007). The tissue level of some polypeptidesincluding aldehyde-dehydrogenase (ADH), chymotrypsin antitryp-




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Table 1Potential diagnostic and prognostic HCC biomarkers.

Potentialbiomarker

Diagnostic application Expression pattern in HCCpatients

References

AFU Diagnosis Upregulation Tangkijvanich et al. (1999)AFU + AFP Diagnosis Upregulation Tangkijvanich et al. (1999)TGF-b1* Prognosis Upregulation Okumoto et al. (2004)VEGF Prognosis Upregulation Poon et al. (2001)AFP* Diagnosis Upregulation Chen et al. (1984) and Farinati et al. (2006)AFP-L3* Diagnosis Upregulation Khien et al. (2001)HSP70 Prognosis Upregulation Decaens and Fartoux (2011)SCCA Diagnosis Upregulation Bertino et al. (2011)GP73 Diagnosis Upregulation Tangkijvanich et al. (1999) and Capurro et al. (2003)GPC3* Diagnosis Upregulation Capurro et al. (2003)FC-GP73 Diagnosis Upregulation Capurro et al. (2003)GGT Diagnosis Upregulation Cui et al. (2003)AFP-mRNA Prognosis and Recurrence Upregulation Matsumura et al. (1994) and Jeng et al. (2004)DCP Early diagnosis and prognosis Upregulation Weitz and Liebman (1993) and Marrero et al. (2003)HCBR2 Prognosis Downregulation Liu et al. (2006)GOLPH2 Tumor aggressiveness Upregulation Riener et al. (2009) and Marrero et al. (2003)HGF Prognosis and disease recurrence NA Osada et al. (2008) and Korn (2001)TSGF Diagnosis complementary to other

markersNA Zhou et al. (2006)

EGFR family* Early recurrence Irregular Ito et al. (2001)miR-21 Diagnosis Upregulation Rfam:miRNA, Ferracin et al. (2010), Ura et al. (2009), Ladeiro et al. (2008) and

Chung et al. (2010)miR-122* Prognosis Downregulation Ladeiro et al. (2008) and Wei et al. (2013)miR-29 Prognosis Downregulation Ura et al. (2009) and Wei et al. (2013)miR-500 Prognosis Downregulation Wei et al. (2013)

NA – Data not available: There is not clear evidence available which suggests that above biomarkers are induced by only virus. But mostly are involved in indirect way ininducing HCC.AFU, a-1-fucosidase; AFP, alpha-fetoprotein; HCC, hepatocellular carcinoma; HBV/HCV, hepatitis B virus/C virus; TGF-b1, transforming growth factor-b1; VEGF, vascularendothelial growth factor; HSP 70, heat shock protein 70; SCCA, squamous cell carcinoma antigen; GP73, golgi protein (Glypican) 73; GGT, c-glutamyl transferase; DCP, des-gamma-carboxy prothrombin; GPC3, glypican-3; FC-GP73, fucosylated GP73; HCBR2, human carbonyl reductase 2; GOLPH2, Golgi phosphoprotein 2; HGF, hepatocytegrowth factor; TSGF, tumor specific growth factor; EGFR, epidermal growth factor receptor; miR, miRNA.

* Marker linked with virus.



27 June 2014

sin, c-reactive proteins have been reported by immunostaining HCCtissue (Hurlimann and Gardiol, 1991). Similarly p53 mutations ischaracteristic in HCC, present 10–20% of tumor characterized isassociated with significant geographical and environmental factors(Hsu et al., 1993). Whereas virus induced HCC causes hepatocyteinjury, chronic inflammation, and accumulation of mutations inthe host genome resulting in chromosomal rearrangements, geneticalteration, activation of oncogenes and inactivation of tumor sup-presser genes (But et al., 2008) and activation of proinflammatorycytokine, interleukins (Cua et al., 2007). Both HBV and HCV viralinfection increase the likelihood of developing liver cancer. It isreported that about half of liver cancer cases are attributable toHBV, and one third of liver cancer cases are attributable to HCV(Sanyal et al., 2010). Though it is not easy to distinguish betweenclinical characters ensuing from hepatitis B and C as well as bothas the cause for PHCC. Therefore better understanding of etiologyof HCC may offer best chance of achievements earlier diagnosisand intervention, which ultimately improve the risk of developingHCC induced through viral infections. Therefore, it is important toidentify actuate and useful biomarkers for early diagnosis of HCCand correct treatment decisions. As we reviewed in this article, sev-eral useful markers have been reported and are summarized inTable 1. It is difficult to find one marker that is both specific and sen-sitive. Thus a combination of at least two or three markers is highlyrecommended for more specific and sensitive diagnosis for HCC.HCC biomarkers AFP-L3 and DCP are intended to be used in in vitrodiagnostic as they are shown to be specific to HCC and their com-bined use aids in early detection of chronic liver disease. Variousstudies have shown the clinical utility of the HCC biomarkers isimproved with the combination of biomarkers (Arii et al., 2010;Choi et al., 2013; Ertle et al., 2013; Nomura et al., 1999;Shimauchi et al., 2000; http://www.wakodiagnostics.com/hccbio-


markers.html). The AFP-L3 and DCP biomarkers are both comple-mentary and are effective in early detection of HCC. The combineduse of these tests is currently available with a single test code as lab-oratories can measure levels of AFP-l3 and DCP with a single serumsample on a single analyzer (Choi et al., 2013; Kagebayashi et al.,2009). Increase in concentration of AFP-L3 isoform indicates PHCCand germ cell tumor. It is more specific than total AFP and otherforms of AFP isoforms in HCC patients (Sato et al., 1993). The testrepresents the ratio of AFP-L3 to total AFP in percentage. Higher lev-els of AFP-L3 values (P10%) have indicated associated with 7-foldrisk of HCC within next 21 months (Liebman et al., 1984). Similarlyelevated DCP (P75 ng/ml) is associated with 5-fold of HCC risk(http://www.wakodiagnostics.com/pivka_dcptest.html).

Similarly a meta-analysis was conducted by Hu B et al., by usingarea under curve (AUC) to evaluate the diagnostic accuracy of com-bination of biomarkers. Combination of AFP+GP73 is better to AFP indiagnosing HCC as well as differentiating HCC patients from non-HCC patients and can be considered as useful diagnosis biomarker(Hu et al., 2013). Squamous cell carcinoma antigen (SCCA) a familyof serine proteases along with total AFP can be potential for combi-national screening leading to an accuracy of 90% (Giannelli andAntonaci, 2006). Though many tumor markers have been reportedin various studies, but none of them are completely optimal. There-fore combinations of two or three HCC biomarkers are recom-mended for highly specific and sensitive diagnosis of HCC.

For decades the most widely used biochemical blood test used todetect liver cancer is detection of alpha fetoprotein (AFP) which ismade by immature fetal liver cells and appears in cancer cells(Benowitz, 2007). Serum AFP levels of more than 400 ng/ml is con-sidered diagnostic but this much value is observed only in very fewcases (Behne and Copur, 2012). Also the false negative rate is quitehigh with early stage HCC. Even in chronic HCC the AFP level found


http://www.wakodiagnostics.com/hccbiomarkers.html

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27 June 2014

to be normal in 15–30% of patients (Singhal et al., 2012). AFP is oftenfound to be elevated for reason other than cancer including liverinjury so it does not necessarily indicate the presence or absenceof liver cancer (Benowitz, 2007). Several biomarkers are in clinicaltrials now but only AFP L3 or fucosylated is FDA approved. Withthe increase of AFP-L3 chance of liver cancer also increases. Cur-rently, alpha-fetoprotein blood test is clinically offered and this testcan also be used in patients already diagnosed with HCC that isavailable at less than 1 US $/test. The AFP level can help in determin-ing if the treatment is effective as the AFP level should go down.Other blood tests such as liver function test, blood clotting tests,tests for viral hepatitis, kidney function test and complete bloodcount test are presently used as they measure levels of certain sub-stances in the blood that determines the function of the liver. Theother two promising biomarker candidates are DCP and GP73(Benowitz, 2007). DCP which is a precursor of a liver protein thathelps in blood clotting. Its level increases in case of liver cancer.DCP is found to be more accurate than AFP in diagnosis of liver can-cer. Other promising candidate is GP73 an early detection bio-marker for liver disease and one of several potential glycoproteinbiomarker for liver disease. Poorly designed studies, lack of diversepopulation studies have hampered the progress of biomarker dis-covery. At present the testing for AFP + PIVKA-II in intervals of3 months is more effective in diagnosing early stages of HCC thanthe 6 months interval of AFP which is normally used (http://www.cancer.gov/clinicaltrials). At the present the screening forHCC in patients with liver cirrhosis is conducted by ultrasoundand measurement of AFP. In this trial the biomarkers AFP- L3 andDCP are calculated in order to receive information about the courseof these biomarkers before the detection of a HCC nodule. Thefutures of developing biomarkers for HCC must describe a consen-sual sorting of HCC with a common terminology based on genomicand well-established molecular information as well as to identifyprognostic markers in HCC and predictors to treatment responseand to develop a prospect plan to further integrate additionalmolecular and clinical information There has been marked advance-ment in the treatment of HCC. However, efficient treatments arelimited to patients with less advanced HCC. The diagnosis of HCCat an early stage is still a must for enhanced prognosis. To addressthis trouble, a variety of screening modalities are used, includingmeasurement of alpha-fetoprotein (AFP) and ultrasonography(US) at regular intervals in high-risk populations. Other markershave been used in some counties, but largely are not commerciallyavailable. MiRNA 122 can be good blood based marker to be devel-oped in the future. Unfortunately, poor sensitivity and specificity ofAFP and the operator-dependency of US limit the value of either testto diagnose early-stage lesions. Current developments in gene-expressing microarrays and proteomics assure even more potentialdiagnostic options to improve the prediction of HCC.

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