**Conditions that Predispose to the Development of HCC: The Role of Tumor Associated Fibroblasts and of microRNA**

C. Avellini, D. Cesselli, A.P. Beltrami, M. Orsaria,

S. Marzinotto, F. Morassi and S. Uzzau

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56951

### **1. Introduction**

The high frequency of cases of hepatocellular carcinoma (HCC) led to vigorous efforts to identify the biological behavior and the pathogenetic mechanisms of this disease. The esti‐ mated 564.000 new cases worldwide and almost the same number of deaths in 2000 make it necessary to understand how to treat and control HCC [1]. This is possible acquiring a deep knowledge of the natural history of the disease, particularly of its initial steps which bring the parenchyma to the first changes and expose the cells to chronic insults, leading to a long standing disease endowed with a high risk of cancer development.

The pathogenesis of HCC is a complex phenomenon-, given the high number of variables and factors involved in liver function and of the possible pathway activation or deregulation during liver disease.

The first valuable step is based upon the death of hepatocytes: different pathological conditions able to induce hepatocyte damage and death will determine recruitment of inflammatory cells and deposition of connective tissue.

On the one hand this fact shows the relatively limited range of liver parenchyma response to various kind of damage. The structure and function of the organ are associated with necroin‐ flammatory, fibrotic and cholestatic changes as the main phenomena which the parenchyma response to damage is able to produce.

On the other hand, this kind of elementary lesions is mostly self-maintaining: in fact, some of the main consequences of hepatocyte destruction and fibrotic parenchymal substitution are

© 2013 Avellini et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. the changes in blood flow and in the composition and function of both extracellular matrix and microenvironment of the liver [1], which heavily influence hepatocyte life and function.

also in HCC. From this point of view the foci of phenotypically altered hepatocytes represent

Conditions that Predispose to the Development of HCC: The Role of Tumor Associated Fibroblasts and of microRNA

http://dx.doi.org/10.5772/56951

175

The foci of altered hepatocytes contain differentiated hepatocytes that have acquired molecular aberrations and changed their metabolic properties [3]: this may allow the progress to malignant phenotype. These cells and their morphological and biochemical phenotype are unstable and, given the absence of uniformity in foci, the clonal origin is partly suggested: at least 8 cytomorphological and cytochemical atypical foci types exist [3]. These premalignant

The main etiological agents of HCC are well known: HBV,HCV and Aflatoxin B1 are consid‐ ered causative agents of about 80% of cases [1]. During last years a lot of genomic aberrations have been identified and the mechanisms of interaction at molecular level between liver cells and etiologic agents in producing gene changes responsible of the development of neoplasia

After the production of foci of altered hepatocytes, the subsequent step, morphologically defined, is represented by the formation of dysplastic foci and dysplastic nodules: in these lesions, probably, molecular changes can take further development with a selection of genetic and epigenetic changes leading to a proliferative advantage for altered hepatocytes and to the involvement of several regulatory pathways in simultaneous disorders favoring the passage

The simple increase in cell proliferation and DNA synthesis associated with increasing

The production of growth factors and hormones (e.g. Insulin, IGF2, TGF alfa, TGF beta, HGF, Transferrin, VEGF), of cytokines and chemokines and the activation or deregulation of different pathways give an essential contribution to the development of neoplasia, but the whole mechanism of the transformation from dysplastic to fully neoplastic lesion is not yet

A crucial element to consider in this context comes from the interaction between the different

For example, hepatocyte proliferation is regulated by cytokines (especially IL-6) produced by Kupffer cells as well as by endothelial cells and hepatic stellate cells. On the other hand, hepatocytes modulate sinusoidal endothelium phenotype and its production of prostaglan‐ dins, endothelin, IL-1 e IL-6 and Hepatocyte Growth Factor [4]. Moreover, the complexity of cross talk between cellular types is relevant in the relationship between hepatocytes and hepatic stellate cells: the production of peptides stimulating hepatic stellate cells proliferation leads to the increased production of cytokines by hepatic stellate cells. These latter are able either to inhibit or to stimulate hepatocyte proliferation [5]. For example, TGF-beta produced by hepatic stellate cells can act as an inhibitor of liver cells replication; moreover, IL-6 response is downregulated by SOCS-3 (suppressor of cytokine signaling 3) with blocking of JAK

enzymatic and metabolic changes is not enough to give rise to carcinogenesis.

the first discrete step, morphologically identifiable, in the way leading to cancer.

changes represent perhaps one of the first effects of viral or environmental damage.

have been studied and often understood [1].

from dysplastic to neoplastic lesion.

cell types present in liver parenchyma.

mediated STAT-3 activation [4, 5].

completely understood.

There is a strict connection between different phenomena concerning the liver in normal and pathological conditions. Chronic inflammation and fibrosis are well known as the main changes leading to neoplasia harboring in the majority of chronic liver diseases; but some insights into the mechanisms and the cellular types which can act either in organ development, or in regenerative and neoplastic processes represent one of the objective of this text. At this regard, a particular role in the relationship between microenvironment and liver cells prolif‐ eration concerns tumor associated fibroblasts (TAFs), considered critical for tumor growth and progression.

Experimental studies in solid tumors other than HCC suggest a possible origin of these cells from mesenchymal stem cells derived from the bone marrow, but in vitro human models lack. These cells exert important functions in cirrhotic and neoplastic liver parenchyma: recently it has been demonstrated the presence in human neoplastic livers of a population of multipotent adult stem cells with properties of tumor associated fibroblasts, while a population of MASCs derived TAFs is already present in cirrhotic, not yet neoplastic parenchyma. Furthermore, mesenchymal stem cells isolated from non-neoplastic and non-cirrhotic livers can acquire a TAF phenotype when grown in a medium conditioned by tumor cell lines [2]

After a brief evaluation of the main factors playing important roles in the pathogenesis and progression of hepatocellular carcinoma (pathways deregulation with respect to morpholog‐ ical changes, molecular factors and pathways cross-talk influencing interplay between different cellular elements, role of extracellular matrix and angiogenesis), the relationship between liver embryogenesis, regeneration and neoplastic growths is considered, inparticular as far as the interplay between parenchymal cells and stromal component in non-neoplastic and neoplastic liver is concerned. Further deeper look is addressed to more recent progresses, which can really offer new important insights in the pathogenesis and treatment of hepato‐ cellular carcinoma: microRNA deregulation and tumor associated fibroblasts. The former show a great potential in targeting a lot of genes involved in the neoplastic process: different tumors in different organs show strict relationships with the effects of groups of miRNA similarly associated for some organs, giving rise to signatures characteristic of neoplastic development, able to predict prognosis and to represent targets for new therapeutic chances. Similar results come from the action of miRNA in hepatocellular carcinoma, where distinct tumor subtypes are associated with different miRNA signature.

Tumor associated fibroblasts represent a population of stromal cells able to influence in crucial ways the parenchymal microenvironment changes which accompany all steps of HCC development, from the origin to progression and diffusion.

#### **1.1. Growth factors and molecular pathways**

With the ongoing of the pathological process the proliferation of liver cells accelerates and monoclonal populations occur: this is the hallmark of all preneoplastic lesions and takes places also in HCC. From this point of view the foci of phenotypically altered hepatocytes represent the first discrete step, morphologically identifiable, in the way leading to cancer.

the changes in blood flow and in the composition and function of both extracellular matrix and microenvironment of the liver [1], which heavily influence hepatocyte life and function.

There is a strict connection between different phenomena concerning the liver in normal and pathological conditions. Chronic inflammation and fibrosis are well known as the main changes leading to neoplasia harboring in the majority of chronic liver diseases; but some insights into the mechanisms and the cellular types which can act either in organ development, or in regenerative and neoplastic processes represent one of the objective of this text. At this regard, a particular role in the relationship between microenvironment and liver cells prolif‐ eration concerns tumor associated fibroblasts (TAFs), considered critical for tumor growth and

Experimental studies in solid tumors other than HCC suggest a possible origin of these cells from mesenchymal stem cells derived from the bone marrow, but in vitro human models lack. These cells exert important functions in cirrhotic and neoplastic liver parenchyma: recently it has been demonstrated the presence in human neoplastic livers of a population of multipotent adult stem cells with properties of tumor associated fibroblasts, while a population of MASCs derived TAFs is already present in cirrhotic, not yet neoplastic parenchyma. Furthermore, mesenchymal stem cells isolated from non-neoplastic and non-cirrhotic livers can acquire a

After a brief evaluation of the main factors playing important roles in the pathogenesis and progression of hepatocellular carcinoma (pathways deregulation with respect to morpholog‐ ical changes, molecular factors and pathways cross-talk influencing interplay between different cellular elements, role of extracellular matrix and angiogenesis), the relationship between liver embryogenesis, regeneration and neoplastic growths is considered, inparticular as far as the interplay between parenchymal cells and stromal component in non-neoplastic and neoplastic liver is concerned. Further deeper look is addressed to more recent progresses, which can really offer new important insights in the pathogenesis and treatment of hepato‐ cellular carcinoma: microRNA deregulation and tumor associated fibroblasts. The former show a great potential in targeting a lot of genes involved in the neoplastic process: different tumors in different organs show strict relationships with the effects of groups of miRNA similarly associated for some organs, giving rise to signatures characteristic of neoplastic development, able to predict prognosis and to represent targets for new therapeutic chances. Similar results come from the action of miRNA in hepatocellular carcinoma, where distinct

Tumor associated fibroblasts represent a population of stromal cells able to influence in crucial ways the parenchymal microenvironment changes which accompany all steps of HCC

With the ongoing of the pathological process the proliferation of liver cells accelerates and monoclonal populations occur: this is the hallmark of all preneoplastic lesions and takes places

TAF phenotype when grown in a medium conditioned by tumor cell lines [2]

tumor subtypes are associated with different miRNA signature.

development, from the origin to progression and diffusion.

**1.1. Growth factors and molecular pathways**

progression.

174 Hepatocellular Carcinoma - Future Outlook

The foci of altered hepatocytes contain differentiated hepatocytes that have acquired molecular aberrations and changed their metabolic properties [3]: this may allow the progress to malignant phenotype. These cells and their morphological and biochemical phenotype are unstable and, given the absence of uniformity in foci, the clonal origin is partly suggested: at least 8 cytomorphological and cytochemical atypical foci types exist [3]. These premalignant changes represent perhaps one of the first effects of viral or environmental damage.

The main etiological agents of HCC are well known: HBV,HCV and Aflatoxin B1 are consid‐ ered causative agents of about 80% of cases [1]. During last years a lot of genomic aberrations have been identified and the mechanisms of interaction at molecular level between liver cells and etiologic agents in producing gene changes responsible of the development of neoplasia have been studied and often understood [1].

After the production of foci of altered hepatocytes, the subsequent step, morphologically defined, is represented by the formation of dysplastic foci and dysplastic nodules: in these lesions, probably, molecular changes can take further development with a selection of genetic and epigenetic changes leading to a proliferative advantage for altered hepatocytes and to the involvement of several regulatory pathways in simultaneous disorders favoring the passage from dysplastic to neoplastic lesion.

The simple increase in cell proliferation and DNA synthesis associated with increasing enzymatic and metabolic changes is not enough to give rise to carcinogenesis.

The production of growth factors and hormones (e.g. Insulin, IGF2, TGF alfa, TGF beta, HGF, Transferrin, VEGF), of cytokines and chemokines and the activation or deregulation of different pathways give an essential contribution to the development of neoplasia, but the whole mechanism of the transformation from dysplastic to fully neoplastic lesion is not yet completely understood.

A crucial element to consider in this context comes from the interaction between the different cell types present in liver parenchyma.

For example, hepatocyte proliferation is regulated by cytokines (especially IL-6) produced by Kupffer cells as well as by endothelial cells and hepatic stellate cells. On the other hand, hepatocytes modulate sinusoidal endothelium phenotype and its production of prostaglan‐ dins, endothelin, IL-1 e IL-6 and Hepatocyte Growth Factor [4]. Moreover, the complexity of cross talk between cellular types is relevant in the relationship between hepatocytes and hepatic stellate cells: the production of peptides stimulating hepatic stellate cells proliferation leads to the increased production of cytokines by hepatic stellate cells. These latter are able either to inhibit or to stimulate hepatocyte proliferation [5]. For example, TGF-beta produced by hepatic stellate cells can act as an inhibitor of liver cells replication; moreover, IL-6 response is downregulated by SOCS-3 (suppressor of cytokine signaling 3) with blocking of JAK mediated STAT-3 activation [4, 5].

Regarding the interaction between hepatocytes and Kupffer cells, growth factors and cytokines activate critical transcription factors, such as AP-1, NFkB, STAT-3, inducing Kupffer cells activation and production of IL-6 thus increasing liver cell DNA synthesis [5].

The proangiogenetic factors exert their influence in particular by activating endothelial cells, which, when activated, loose interendothelial cells contacts and breakdown the surrounding basement membrane and extracellular matrix: the associated phenomena of proliferation and migration of endothelial cells are widened by further secretion of angiogenic factors,former‐ lysequestered in perivascular extracellular matrix. The secretion of protease induces the release

Conditions that Predispose to the Development of HCC: The Role of Tumor Associated Fibroblasts and of microRNA

http://dx.doi.org/10.5772/56951

177

Endothelial cells proliferation and migration is followed by the tendency to assemble in tubular structures, with subsequent basement membrane material production and peri‐ cytes (supportive vascular smooth muscle cells) recruitment through the action of PDGF beta: these newly formed vascular channels nevertheless show irregular and variable diameter, with abnormal branching pattern, incomplete structure of basement membrane and only partialpericytes cover, which represent the main characteristics of new vascular channels associated with the development of neoplastic growth at the beginning. The process go on when phenomena ofhypoxia in the central area of the tumoror some viral component if present ( for example HBX protein ) stimulate the production of Hipoxia Inducible Factor-1alfa, followed by the action of VEGF glycoproteins (A,B,C,D and placental growth factor) on corresponding receptors (flt1 and flk1) on endothelial cells. It has been demonstrated that Trans arterial chemoembolization (TACE) for hepatocellular carcinoma treatment is followed by parallel increase of VEGF expression related to microvascular density and hypoxia [6, 7], which confirms the relationship between ischemia and reac‐

Angiogenic stimuli produce different effects in normal, cirrhotic and neoplastic liver paren‐ chyma:within normal and regenerating tissue new functional sinusoids appear, while in

The high number of complex interplays, here rapidly summarized, underlines the crucial connection between parenchymal cells, non-parenchymal cells and extracellular matrix, including vascular component. Again this phenomenon-show significantsimilarities between

**2. Liver development, regeneration, neoplasia: The role of stem cells**

It has been recognized the existence, in the liver, of a strict connection between development, regeneration, and carcinogenesis [8, 9]. As a consequence, researchers are trying to dissect the molecular mechanisms regulating liver homeostasis, the comprehension of which could open the way to new targeted therapies for liver regeneration, liver cirrhosis and primary liver

Liver development involves complex mechanisms. Multipotent tissue specific progenitor cells, derived from blastocyst inner cell mass stem cells, give rise to the different organs [8]. Specifically, fibroblast growth factor (FGF) from the cardiac mesoderm, through a coordination of signaling with bone morphogenic proteins from the septum transversum mesenchyme,

chronic liver disease capillarized vascular structures take place [7].

different physiological and pathological events involving the liver.

of free vascular endothelial growth factor which stimulates endothelial cells [6, 7].

tive neoangiogenesis.

cancers [8, 9].

#### **1.2. Extracellular matrix**

The site where the described interactions take place is not a silent structure away from any biochemical activity: the relationship between hepatocytes and extracellular matrix is partic‐ ularly active and crucial [3].

A lot of adhesion molecules and receptor mediating cell-matrix binding are present.

Extracellular matrix acts as a reservoir and presenter of cell growth factors and cytokines; it undergoes a rapid turnover and significant modifications, often induced by liver parenchymal and non-parenchymal cells, through the production of matrix-degrading enzymes, such as metalloproteinases, and their inhibitors which control the extracellular matrix degradation. Soluble mediators able to influence hepatic stellate cells and Kupffer cells induce the produc‐ tion of proteinases which may initiate the process of matrix degradation. Kupffer cells adhesion on endothelial cells via CD4 and ICAM-1 produce hepatic stellate cells stimulating factors: again, hepatic stellate cells, modulated by hepatocytes, endothelial cells, Kupffer cells, platelets and inflammatory cells play a role in fibrogenesis and liver morphogenesis.

When extracellular matrix at sinusoidal subendothelial level undergoes some kind of disrup‐ tion, hepatocytes can lose their differentiated function and morphofunctional changes take place: altered porosity in sinusoidal barrier and impaired movement of solutes and macro‐ molecules into and out of Disse spaces favor fibrogenetic progression, with further functional impairment. In particular, extracellular matrix turnover and degradation are under the control of different factors, either at extracellular (metalloproteinases) or intracellular level (lysoso‐ malcathepsins) [3].

Collagenases acting on fibrillar collagen, stromelysins (degrading collagen IV, gelatin, laminin, fibronectin), gelatinase, neutrophilic collagenase ( acting on collagen I and III) and so on parallel the activity of molecules expressed by hepatocytes and hepatic stellate cells, function‐ ing as inhibitors of metalloproteinases (tissue inhibitors of metalloproteinases 1 and 2 and alfa 2 macroglobulin), with a regulation of relative gene expression by same cytokines and growth factors as metalloproteinases [3].

Interstitial collagenase activity is increased in liver fibrosis, mainly during the early develop‐ ment of extensive lesions, but diminishes with advanced cirrhosis.

#### **1.3. Angiogenesis**

Fibrogenetic progression with Disse spaces fibrosis and sinusoidal capillarization induces great impairment particularly in hepatocellular secretion of proteins (albumin, clotting factors, lipoproteins), associated with dramatic changes in fluid dynamics [3].

Genetic changes and local hypoxia may lead to secretion of soluble angiogenic factors, with a complex interplay between cells, basal membranes and pro-or antiangiogenic factors.

The proangiogenetic factors exert their influence in particular by activating endothelial cells, which, when activated, loose interendothelial cells contacts and breakdown the surrounding basement membrane and extracellular matrix: the associated phenomena of proliferation and migration of endothelial cells are widened by further secretion of angiogenic factors,former‐ lysequestered in perivascular extracellular matrix. The secretion of protease induces the release of free vascular endothelial growth factor which stimulates endothelial cells [6, 7].

Regarding the interaction between hepatocytes and Kupffer cells, growth factors and cytokines activate critical transcription factors, such as AP-1, NFkB, STAT-3, inducing Kupffer cells

The site where the described interactions take place is not a silent structure away from any biochemical activity: the relationship between hepatocytes and extracellular matrix is partic‐

Extracellular matrix acts as a reservoir and presenter of cell growth factors and cytokines; it undergoes a rapid turnover and significant modifications, often induced by liver parenchymal and non-parenchymal cells, through the production of matrix-degrading enzymes, such as metalloproteinases, and their inhibitors which control the extracellular matrix degradation. Soluble mediators able to influence hepatic stellate cells and Kupffer cells induce the produc‐ tion of proteinases which may initiate the process of matrix degradation. Kupffer cells adhesion on endothelial cells via CD4 and ICAM-1 produce hepatic stellate cells stimulating factors: again, hepatic stellate cells, modulated by hepatocytes, endothelial cells, Kupffer cells, platelets

When extracellular matrix at sinusoidal subendothelial level undergoes some kind of disrup‐ tion, hepatocytes can lose their differentiated function and morphofunctional changes take place: altered porosity in sinusoidal barrier and impaired movement of solutes and macro‐ molecules into and out of Disse spaces favor fibrogenetic progression, with further functional impairment. In particular, extracellular matrix turnover and degradation are under the control of different factors, either at extracellular (metalloproteinases) or intracellular level (lysoso‐

Collagenases acting on fibrillar collagen, stromelysins (degrading collagen IV, gelatin, laminin, fibronectin), gelatinase, neutrophilic collagenase ( acting on collagen I and III) and so on parallel the activity of molecules expressed by hepatocytes and hepatic stellate cells, function‐ ing as inhibitors of metalloproteinases (tissue inhibitors of metalloproteinases 1 and 2 and alfa 2 macroglobulin), with a regulation of relative gene expression by same cytokines and growth

Interstitial collagenase activity is increased in liver fibrosis, mainly during the early develop‐

Fibrogenetic progression with Disse spaces fibrosis and sinusoidal capillarization induces great impairment particularly in hepatocellular secretion of proteins (albumin, clotting factors,

Genetic changes and local hypoxia may lead to secretion of soluble angiogenic factors, with a

complex interplay between cells, basal membranes and pro-or antiangiogenic factors.

ment of extensive lesions, but diminishes with advanced cirrhosis.

lipoproteins), associated with dramatic changes in fluid dynamics [3].

A lot of adhesion molecules and receptor mediating cell-matrix binding are present.

and inflammatory cells play a role in fibrogenesis and liver morphogenesis.

activation and production of IL-6 thus increasing liver cell DNA synthesis [5].

**1.2. Extracellular matrix**

176 Hepatocellular Carcinoma - Future Outlook

ularly active and crucial [3].

malcathepsins) [3].

**1.3. Angiogenesis**

factors as metalloproteinases [3].

Endothelial cells proliferation and migration is followed by the tendency to assemble in tubular structures, with subsequent basement membrane material production and peri‐ cytes (supportive vascular smooth muscle cells) recruitment through the action of PDGF beta: these newly formed vascular channels nevertheless show irregular and variable diameter, with abnormal branching pattern, incomplete structure of basement membrane and only partialpericytes cover, which represent the main characteristics of new vascular channels associated with the development of neoplastic growth at the beginning. The process go on when phenomena ofhypoxia in the central area of the tumoror some viral component if present ( for example HBX protein ) stimulate the production of Hipoxia Inducible Factor-1alfa, followed by the action of VEGF glycoproteins (A,B,C,D and placental growth factor) on corresponding receptors (flt1 and flk1) on endothelial cells. It has been demonstrated that Trans arterial chemoembolization (TACE) for hepatocellular carcinoma treatment is followed by parallel increase of VEGF expression related to microvascular density and hypoxia [6, 7], which confirms the relationship between ischemia and reac‐ tive neoangiogenesis.

Angiogenic stimuli produce different effects in normal, cirrhotic and neoplastic liver paren‐ chyma:within normal and regenerating tissue new functional sinusoids appear, while in chronic liver disease capillarized vascular structures take place [7].

The high number of complex interplays, here rapidly summarized, underlines the crucial connection between parenchymal cells, non-parenchymal cells and extracellular matrix, including vascular component. Again this phenomenon-show significantsimilarities between different physiological and pathological events involving the liver.

### **2. Liver development, regeneration, neoplasia: The role of stem cells**

It has been recognized the existence, in the liver, of a strict connection between development, regeneration, and carcinogenesis [8, 9]. As a consequence, researchers are trying to dissect the molecular mechanisms regulating liver homeostasis, the comprehension of which could open the way to new targeted therapies for liver regeneration, liver cirrhosis and primary liver cancers [8, 9].

Liver development involves complex mechanisms. Multipotent tissue specific progenitor cells, derived from blastocyst inner cell mass stem cells, give rise to the different organs [8]. Specifically, fibroblast growth factor (FGF) from the cardiac mesoderm, through a coordination of signaling with bone morphogenic proteins from the septum transversum mesenchyme, influences the hepatic induction [10-14]. In fact, committed foregut endoderm elements, through WNT signaling, promote liver bud emergence [15-17]: hepatoblasts invade the septum transversum mesenchyme to give rise to liver bud and proliferate under the influence of mesenchymal cells derived cytokines and growth factors, such as FGF, HGF and TGF beta [18-21] and of neighboring endothelial cells signaling.

Chronic inflammatory response, growth factors and DNA damaging agents (including ROS) also play a role [25, 26], favoring the involvement of progenitor cells in regenerative / prolif‐

Conditions that Predispose to the Development of HCC: The Role of Tumor Associated Fibroblasts and of microRNA

http://dx.doi.org/10.5772/56951

179

Type and duration of exposure to ROS, increasing after cellular stress, play a pivotal role in choosing the direction of cells destiny. An acute and transient increase in ROS is necessary for proto-oncogenes and growth factors to induce cellular proliferation [24]. Interestingly, similarly toregenerative response, with H-RAS mediated proliferative activity, malignant transformation also requires K-RAS proto-oncogene activation, inducing ERK 1-2 activation [24]. Surviving hepatocytes surveillance could therefore be overwhelmed by stress induced

On the one hand, normal livers, which undergone experimental hepatectomy, show a regen‐ erative, mitotic response starting from adult hepatocytes. On the other hand, in the presence of a chronic parenchymal damage, the response isbased upon progenitor cells, with molecular

A part from experimental parenchymal damage, chronic ethanol consumption generally increases the death rate of mature hepatocytes. This stimulates a compensatory regenerative response to preserve normal liver mass and function. Nevertheless, most of the mature hepatocytes that survive in alcohol damaged livers are replicatively senescent and hence incapable of proliferating to replace their dead neighbors, given the chronic alcohol exposure which inhibits the induction of DNA synthesis. Hence, regeneration of alcohol damaged livers involves the expansion and differentiation of facultative liver progenitor cells [24]. This regenerative mechanism requires lengthening the time needed for liver mass reconstitution,

On the whole, the reparative processes take advantage of two principal strategies of defense, depending on the extent of the damage [8]. While mild injury is mainly repaired through compensatory hyperplasia of hepatocytes, severe damage implies the activation of a liver progenitor cell compartment [27]. In both cases, other non-parenchymal cells (stellate cells, vascular and biliary cells) proliferate as soon as hepatocytes starts to [27]and cooperate to restore morphofunctional competent liver tissue. In rodent experimental models with chronic

HGF and EGF promote the upregulation of proliferation rate and expansion of oval cells, while TGF-beta exerts the opposite effect on oval cells [28-31], with an upregulation of the WNT-Beta Catenin pathway in acute and chronic liver injury experimental models [8]. These data again suggest symmetry between fetal development and regenerative mechanisms. Molecular mechanisms involved in fetal development, when identified, can allow to target pathways connected with parenchymal damage: tyrosine kinase inhibitors, for instance, are able to inhibit progenitor cell response, liver fibrosis and liver cancer development in a mouse model of chronic liver injury [32]; C-kit inhibition by imatinibmesylate attenuates progenitor cell

erative activity and therefore probably also favoring tumor development.

signaling patterns that suggest a recapitulation of fetal development [8].

proliferative stimuli associated to malignant transformation.

due to the time necessary for differentiation of progenitor cells.

expansion and inhibits liver tumor formation in mice [32].

liver injury.

The hepatoblasts show bipotential characteristic and specific pathways induce their differen‐ tiation into hepatocytes or biliary epithelium, respectively. Some studies suggest the ductal plate as the site of fetal liver progenitor cells, with 4 hypothetical anatomic compartments [22]: hepatocytes which meet the canal of Hering, pankeratin positive cells in the canal of Hering, intraductalcholagiocytes, peribiliary" null" cells.

After injury, liver regeneration processes are strictly related to the extent of the loss of liver parenchyma [8] Specifically, after experimental partial hepatectomy, mass restoration is mainly due to mitotic division of mature liver cells [23], while when this regenerative capacity is overwhelmed by massive parenchymal loss, for example in chronic liver disease, mass regeneration implies the activation of a liver progenitor cell compartment [22]. The molecular pathways activated in this latter case suggest a recapitulation of the fetal development [8]. In fact progenitor cells phenotypes and antigenic profiling are similar to fetal liver: these cells are located at the canal of Hering; the presence of label retaining cells within the iuxta portal, proximal biliary tree (slow cycling cells which retain Bromodeoxyuridin (BRDU) label for 8 weeks after experimental injury related cell division) confirm this site as a possible stem cell niche [22]. Similar observations concern peribiliary hepatocytes, mostly identified as BRDU positive very early in post-injury period, at variance with mid-acinar and central acinar hepatocytes, involved in rapid turnover for regenerative response to the loss of parenchyma. On the other hand, intraductalcholangiocytes and peribiliary "null" cells are less likely to take a role in the niche population, due also to the difficult evaluation of their relationship with neighbor liver parenchyma [22].

Liver regeneration takes places within some days or few weeks from the insult. After experimental partial hepatectomy, inflammatory cells produce TNF-alfa which damages surviving cells, but induces DNA synthesis and adult liver cells proliferation; this mecha‐ nism needs the presence of a specific receptor on hepatocytes (Tumor necrosis factor Receptor 1). TNFR1-null mice can nevertheless grow to adulthood, demonstrating the possible development also in the absence of this interaction TNF alfa-TNFR1. This is simply one out of several mechanisms (such as the action of drugs or hormones) which increase hepatocyte proliferation, while interaction with TNF is not a unique way for liver develop‐ ment [24]: although TNF production and Glutation (GSH) synthesis represent rapid reaction to experimental hepatectomy, TNF simply activates different factors (such as NFKB and SEK-1, a stress related kinase) that permit hepatocytes to survive to TNF exposure. "Surviving hepatocytes are then surveyed for damage and repaired or deleted" [24]: this is a defensive, initial mechanism that, together with antiapoptotic and antioxidant factors, makepossible subsequent parenchymal regeneration. All these observations show a complex picture of stress regulated intracellular signals that allow adult hepatocytes to proliferate, to survive without proliferation or to dye.

Chronic inflammatory response, growth factors and DNA damaging agents (including ROS) also play a role [25, 26], favoring the involvement of progenitor cells in regenerative / prolif‐ erative activity and therefore probably also favoring tumor development.

influences the hepatic induction [10-14]. In fact, committed foregut endoderm elements, through WNT signaling, promote liver bud emergence [15-17]: hepatoblasts invade the septum transversum mesenchyme to give rise to liver bud and proliferate under the influence of mesenchymal cells derived cytokines and growth factors, such as FGF, HGF and TGF beta

The hepatoblasts show bipotential characteristic and specific pathways induce their differen‐ tiation into hepatocytes or biliary epithelium, respectively. Some studies suggest the ductal plate as the site of fetal liver progenitor cells, with 4 hypothetical anatomic compartments [22]: hepatocytes which meet the canal of Hering, pankeratin positive cells in the canal of Hering,

After injury, liver regeneration processes are strictly related to the extent of the loss of liver parenchyma [8] Specifically, after experimental partial hepatectomy, mass restoration is mainly due to mitotic division of mature liver cells [23], while when this regenerative capacity is overwhelmed by massive parenchymal loss, for example in chronic liver disease, mass regeneration implies the activation of a liver progenitor cell compartment [22]. The molecular pathways activated in this latter case suggest a recapitulation of the fetal development [8]. In fact progenitor cells phenotypes and antigenic profiling are similar to fetal liver: these cells are located at the canal of Hering; the presence of label retaining cells within the iuxta portal, proximal biliary tree (slow cycling cells which retain Bromodeoxyuridin (BRDU) label for 8 weeks after experimental injury related cell division) confirm this site as a possible stem cell niche [22]. Similar observations concern peribiliary hepatocytes, mostly identified as BRDU positive very early in post-injury period, at variance with mid-acinar and central acinar hepatocytes, involved in rapid turnover for regenerative response to the loss of parenchyma. On the other hand, intraductalcholangiocytes and peribiliary "null" cells are less likely to take a role in the niche population, due also to the difficult evaluation of their relationship with

Liver regeneration takes places within some days or few weeks from the insult. After experimental partial hepatectomy, inflammatory cells produce TNF-alfa which damages surviving cells, but induces DNA synthesis and adult liver cells proliferation; this mecha‐ nism needs the presence of a specific receptor on hepatocytes (Tumor necrosis factor Receptor 1). TNFR1-null mice can nevertheless grow to adulthood, demonstrating the possible development also in the absence of this interaction TNF alfa-TNFR1. This is simply one out of several mechanisms (such as the action of drugs or hormones) which increase hepatocyte proliferation, while interaction with TNF is not a unique way for liver develop‐ ment [24]: although TNF production and Glutation (GSH) synthesis represent rapid reaction to experimental hepatectomy, TNF simply activates different factors (such as NFKB and SEK-1, a stress related kinase) that permit hepatocytes to survive to TNF exposure. "Surviving hepatocytes are then surveyed for damage and repaired or deleted" [24]: this is a defensive, initial mechanism that, together with antiapoptotic and antioxidant factors, makepossible subsequent parenchymal regeneration. All these observations show a complex picture of stress regulated intracellular signals that allow adult hepatocytes to proliferate,

[18-21] and of neighboring endothelial cells signaling.

178 Hepatocellular Carcinoma - Future Outlook

intraductalcholagiocytes, peribiliary" null" cells.

neighbor liver parenchyma [22].

to survive without proliferation or to dye.

Type and duration of exposure to ROS, increasing after cellular stress, play a pivotal role in choosing the direction of cells destiny. An acute and transient increase in ROS is necessary for proto-oncogenes and growth factors to induce cellular proliferation [24]. Interestingly, similarly toregenerative response, with H-RAS mediated proliferative activity, malignant transformation also requires K-RAS proto-oncogene activation, inducing ERK 1-2 activation [24]. Surviving hepatocytes surveillance could therefore be overwhelmed by stress induced proliferative stimuli associated to malignant transformation.

On the one hand, normal livers, which undergone experimental hepatectomy, show a regen‐ erative, mitotic response starting from adult hepatocytes. On the other hand, in the presence of a chronic parenchymal damage, the response isbased upon progenitor cells, with molecular signaling patterns that suggest a recapitulation of fetal development [8].

A part from experimental parenchymal damage, chronic ethanol consumption generally increases the death rate of mature hepatocytes. This stimulates a compensatory regenerative response to preserve normal liver mass and function. Nevertheless, most of the mature hepatocytes that survive in alcohol damaged livers are replicatively senescent and hence incapable of proliferating to replace their dead neighbors, given the chronic alcohol exposure which inhibits the induction of DNA synthesis. Hence, regeneration of alcohol damaged livers involves the expansion and differentiation of facultative liver progenitor cells [24]. This regenerative mechanism requires lengthening the time needed for liver mass reconstitution, due to the time necessary for differentiation of progenitor cells.

On the whole, the reparative processes take advantage of two principal strategies of defense, depending on the extent of the damage [8]. While mild injury is mainly repaired through compensatory hyperplasia of hepatocytes, severe damage implies the activation of a liver progenitor cell compartment [27]. In both cases, other non-parenchymal cells (stellate cells, vascular and biliary cells) proliferate as soon as hepatocytes starts to [27]and cooperate to restore morphofunctional competent liver tissue. In rodent experimental models with chronic liver injury.

HGF and EGF promote the upregulation of proliferation rate and expansion of oval cells, while TGF-beta exerts the opposite effect on oval cells [28-31], with an upregulation of the WNT-Beta Catenin pathway in acute and chronic liver injury experimental models [8]. These data again suggest symmetry between fetal development and regenerative mechanisms. Molecular mechanisms involved in fetal development, when identified, can allow to target pathways connected with parenchymal damage: tyrosine kinase inhibitors, for instance, are able to inhibit progenitor cell response, liver fibrosis and liver cancer development in a mouse model of chronic liver injury [32]; C-kit inhibition by imatinibmesylate attenuates progenitor cell expansion and inhibits liver tumor formation in mice [32].

Therefore, the presence of pathways playing a role either in fetal development or in regener‐ ative but also in neoplastic phenomena opens important perspectives: three distinct cell lineages can be found in the liver,susceptible of neoplastic transformation: mature hepatocytes, small proliferating hepatocytes and stem cells [33, 34].

"One possibility is that HLSC may represent a mesenchymal population modified by the influence of the local environment, reflecting the importance of the niche in establishing the

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181

The "tumor stroma" can be considered as "normal wound healing gone awry [43], able to interact through paracrine and juxtacrine pathways with the tumor stem cells that are influenced by the microenvironment in which they are endowed, with a direct role of micro‐ environment cells in determining the malignant phenotype [44-52]. In summary, fetal devel‐ opment, regeneration and carcinogenetic processes show some point of similarity in molecular pathways and mechanisms, partly recapitulatedin chronic parenchymal liver damage evolv‐

Starting from the concept of chronic liver disease development, with progressive liver cell destruction, reactive fibrosis and general parenchymal organization disarray, it is possible a

These phenomena are all present since the beginning of liver damage, when liver cells are involved by viral or toxic damage and fibrous tissue deposition replace the destroyed cells. This is the beginning of fibrosis development, firstly at sub-sinusoidal level, then within the

Importantly, evolution of liver fibrosis and activation of hepatic stellate cells share some pathways [4]. In fact, the adhesion molecules and receptors mediating cell-extracellular matrix binding and growth factors and cytokines present in ECM influence matrix turnover and modifications and, together with liver cells, endothelium, Kupffer cells and inflammatory cells, modulate hepatic stellate cells. These latter express Integrins, mediating cell adhesion to fibronectin and collagens and modulating metalloproteinases: integrins activate intracellular signaling pathways in response to ECM protein they recognize, influencing hepatocytes

As a consequence, any kind of liver parenchymal damage, though mild, induces a regenerative

Liver cells, ductular cells, stromal cells and perisinusoidal cells all undergo regenerative phenomena: it is a complex sequence associated with progressive collagen deposition with a healing role, involving subsinusoidalDisse spaces, periportal and perilaminarinterstitium, with ECM and parenchymal cells activation and proliferation and with ECM synthesis by stellate cells, fibroblasts and, in some cases, parenchymal cells. In fact, in this context, epithelialmesenchymal transition and mesenchymal-epithelial transition play a role, with different tissue component phenotypic changes, significantly involving parenchymal structure and

If chronic inflammation with long standing tissue damage represents a risk condition for neoplastic disease, cirrhosis is the typical predisposing setting for hepatocellular carcinoma. In fact, cirrhosis is characterized by a microenvironment possibly favoring HCC onset: most

cases of hepatocellular carcinoma are linked to the presence of cirrhosis [54, 55].

damaged areas, often associated, in more advanced stages, with a ductular reaction.

phenotype of MSC" [42].

ing to advanced stages.

differentiation.

function [53].

better comprehension of the whole changes.

liver cell response, starting from periportal liver cells [25].

Specifically, hepatocellular carcinoma might result from dedifferentiation of mature hepato‐ cytes, from activation of oval cells, from arrested differentiation of tissue based stem cells, the latter being an expression of blocked ontogeny, linked to hepatocarcinogenesis. Evidence of the fact that hepatocarcinogenesis partly recapitulates fetal development comes from some observations: both progenitor and fetal cells are self-renewing, with heterogeneous progeny and limitless division; bipotent cells, with hepatocytic and cholangiocytic potential are present in fetal livers and have been isolated in a number of HCC cell lines; finally, the strict relation‐ ship between tumor cells and fetal program is testified by the fact that HCC cell lines (e.g. Huh1, Huh 7, Hep 3b cell lines) share with fetal liver progenitors several oncofetal markers and that HCC characterized by a gene expression profile similar to fetal hepatoblasts have a poorer prognosis than HCC with an adult-type genomic profile [35-39].

According to the possible origin from progenitor cells or from dedifferentiated adult cells, HCC could assume different phenotypes and be linked to the activation of different pathways, associated to variable parenchymal morphological changes. For instance, significantly fewer HCCs expressing CK7 or CK19 show nuclear beta Catenin expression. The beta catenin pathway could preferentially involve "mature" hepatocytes versus less "mature" (progenitor) phenotype cells, with CK7 and CK19 expression,associated withless advanced fibrosis in nontumoral parenchyma [40].

CK19 expression in HCC can change with different expression of beta catenin: decreased fibrosis degree in the non-tumoral parenchyma parallels reduced nuclear beta catenin expression [25].

Regarding oval cells, many of the compounds that induce their proliferation are DNAdamaging agents or carcinogens, therefore oval cells can be considered as potential precan‐ cerous cells [41]. Oval cells activation with "ductular reaction" represent the expansion of a transit amplifyingcell compartment of small biliary bipotent cells [25].

In cirrhosis, hepatocytes are characterized by senescence determined by telomere shortening, while mesenchymal cells (e.g. endothelial cells and stellate cells) seem not to be affected by replicative senescence [25]. In the hepatocytes this latter can be the result of an enhanced proliferation rate that can persist for 20-30 years of chronic liver disease. It is possible to hypothesize that the parallel proliferative activity of progenitor cells and mesenchymal cells allows an influence of the latter elements on the progenitor cells carcinogenic evolution: it is known that sinusoidal lining cells like hepatic stellate cells proliferate in close anatomical relationship with progenitor cells and are able to produce growth factors for which progenitor cells have the receptors, suggesting an interaction between these cell compartments [26].

"A characteristic of stem cells is to survive to toxic and hypoxic stimuli due to their low cell cycling ".

"One possibility is that HLSC may represent a mesenchymal population modified by the influence of the local environment, reflecting the importance of the niche in establishing the phenotype of MSC" [42].

Therefore, the presence of pathways playing a role either in fetal development or in regener‐ ative but also in neoplastic phenomena opens important perspectives: three distinct cell lineages can be found in the liver,susceptible of neoplastic transformation: mature hepatocytes,

Specifically, hepatocellular carcinoma might result from dedifferentiation of mature hepato‐ cytes, from activation of oval cells, from arrested differentiation of tissue based stem cells, the latter being an expression of blocked ontogeny, linked to hepatocarcinogenesis. Evidence of the fact that hepatocarcinogenesis partly recapitulates fetal development comes from some observations: both progenitor and fetal cells are self-renewing, with heterogeneous progeny and limitless division; bipotent cells, with hepatocytic and cholangiocytic potential are present in fetal livers and have been isolated in a number of HCC cell lines; finally, the strict relation‐ ship between tumor cells and fetal program is testified by the fact that HCC cell lines (e.g. Huh1, Huh 7, Hep 3b cell lines) share with fetal liver progenitors several oncofetal markers and that HCC characterized by a gene expression profile similar to fetal hepatoblasts have a

According to the possible origin from progenitor cells or from dedifferentiated adult cells, HCC could assume different phenotypes and be linked to the activation of different pathways, associated to variable parenchymal morphological changes. For instance, significantly fewer HCCs expressing CK7 or CK19 show nuclear beta Catenin expression. The beta catenin pathway could preferentially involve "mature" hepatocytes versus less "mature" (progenitor) phenotype cells, with CK7 and CK19 expression,associated withless advanced fibrosis in non-

CK19 expression in HCC can change with different expression of beta catenin: decreased fibrosis degree in the non-tumoral parenchyma parallels reduced nuclear beta catenin

Regarding oval cells, many of the compounds that induce their proliferation are DNAdamaging agents or carcinogens, therefore oval cells can be considered as potential precan‐ cerous cells [41]. Oval cells activation with "ductular reaction" represent the expansion of a

In cirrhosis, hepatocytes are characterized by senescence determined by telomere shortening, while mesenchymal cells (e.g. endothelial cells and stellate cells) seem not to be affected by replicative senescence [25]. In the hepatocytes this latter can be the result of an enhanced proliferation rate that can persist for 20-30 years of chronic liver disease. It is possible to hypothesize that the parallel proliferative activity of progenitor cells and mesenchymal cells allows an influence of the latter elements on the progenitor cells carcinogenic evolution: it is known that sinusoidal lining cells like hepatic stellate cells proliferate in close anatomical relationship with progenitor cells and are able to produce growth factors for which progenitor cells have the receptors, suggesting an interaction between these cell compartments [26].

"A characteristic of stem cells is to survive to toxic and hypoxic stimuli due to their low cell

small proliferating hepatocytes and stem cells [33, 34].

180 Hepatocellular Carcinoma - Future Outlook

tumoral parenchyma [40].

expression [25].

cycling ".

poorer prognosis than HCC with an adult-type genomic profile [35-39].

transit amplifyingcell compartment of small biliary bipotent cells [25].

The "tumor stroma" can be considered as "normal wound healing gone awry [43], able to interact through paracrine and juxtacrine pathways with the tumor stem cells that are influenced by the microenvironment in which they are endowed, with a direct role of micro‐ environment cells in determining the malignant phenotype [44-52]. In summary, fetal devel‐ opment, regeneration and carcinogenetic processes show some point of similarity in molecular pathways and mechanisms, partly recapitulatedin chronic parenchymal liver damage evolv‐ ing to advanced stages.

Starting from the concept of chronic liver disease development, with progressive liver cell destruction, reactive fibrosis and general parenchymal organization disarray, it is possible a better comprehension of the whole changes.

These phenomena are all present since the beginning of liver damage, when liver cells are involved by viral or toxic damage and fibrous tissue deposition replace the destroyed cells. This is the beginning of fibrosis development, firstly at sub-sinusoidal level, then within the damaged areas, often associated, in more advanced stages, with a ductular reaction.

Importantly, evolution of liver fibrosis and activation of hepatic stellate cells share some pathways [4]. In fact, the adhesion molecules and receptors mediating cell-extracellular matrix binding and growth factors and cytokines present in ECM influence matrix turnover and modifications and, together with liver cells, endothelium, Kupffer cells and inflammatory cells, modulate hepatic stellate cells. These latter express Integrins, mediating cell adhesion to fibronectin and collagens and modulating metalloproteinases: integrins activate intracellular signaling pathways in response to ECM protein they recognize, influencing hepatocytes differentiation.

As a consequence, any kind of liver parenchymal damage, though mild, induces a regenerative liver cell response, starting from periportal liver cells [25].

Liver cells, ductular cells, stromal cells and perisinusoidal cells all undergo regenerative phenomena: it is a complex sequence associated with progressive collagen deposition with a healing role, involving subsinusoidalDisse spaces, periportal and perilaminarinterstitium, with ECM and parenchymal cells activation and proliferation and with ECM synthesis by stellate cells, fibroblasts and, in some cases, parenchymal cells. In fact, in this context, epithelialmesenchymal transition and mesenchymal-epithelial transition play a role, with different tissue component phenotypic changes, significantly involving parenchymal structure and function [53].

If chronic inflammation with long standing tissue damage represents a risk condition for neoplastic disease, cirrhosis is the typical predisposing setting for hepatocellular carcinoma. In fact, cirrhosis is characterized by a microenvironment possibly favoring HCC onset: most cases of hepatocellular carcinoma are linked to the presence of cirrhosis [54, 55].

### **3. Molecular changes in the development of HCC: The novel role of miRNA**

Many experimental evidences support the relationship between fragile sites and DNA instability in cancerous cells. In fact, fragile sites are preferential sites for chromatids exchang‐ es, translocations, deletions, gene amplification or integrations of associated viral sequences,

Conditions that Predispose to the Development of HCC: The Role of Tumor Associated Fibroblasts and of microRNA

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183

Besides the association with fragile sites, microRNA genes may be involved in tumorigenesis process through other mechanisms, such as point mutation, deletion, amplification, translo‐ cation or epigenetic modifications [61]. Most known microRNA have been identified within genomic cancer-associated regions: minimal regions of loss of heterozigosity, where often oncosuppressor genes are present or minimal region of amplification, where often oncogenes have been identified [62]. This strongly suggest that miRNAgenes may behave either as oncogenes or as tumor suppressor genes and, in particular, the same miRNA may behave in different way on the basis of the kind of alteration, cellular type or transcriptional/post-

miRNA deregulation can also be the consequence of epigenetic changes, In fact, an extensive genomic analysis of miRNA-coding gene sequences showed that about 50% of these loci are CpG island associated [62] makingpossible that an alteration in DNA methylation/acethylation

Regarding the changes in miRNA expression in different tumors, the Volinia study [64], performed a microarray analysis of 20 different miRNAs in 540 specimen obtained from different tumor types (lung, breast, stomach, prostate, colon, pancreas) identifying a miRNA signature common to some different solid tumors). The value of this study, find a confirma‐ tion in the similar grouping, between different tumors, of the miRNA expression signa‐ ture: this suggest a common mechanism of involvement of miRNA in human carcinogenesis. Prostate, colon, stomach, pancreas show mostly similar signature among them, whereas lung and breast are represented by a fairly different signature. Furthermore, within the signature, some miRNA have been found whose association with other human tumors was already known: among them there are miR-17-5p, miR-20a, miR-21, miR-92, miR-106a, e miR-155, and molecular targets of these miRNA are significantly rich in tumor suppres‐ sor genes and oncogenes. These data suggest the crucial involvement of miRNA in the pathogenesis of solid tumors and confirm the function of miRNA as dominant and recessive tumoral genes. Furthermore, it has been shown the role of miRNA not only in the early phases of primitive tumors development, but also during the progression and the metastat‐ ic diffusion of the neoplastic disease. In fact, many experimental evidences show miRNA involvement in the regulation of biological process leading to the acquisition of metastat‐ ic potential, such as adhesion, invasion, migration, epithelial-to-mesenchymal transition and

Among miRNAs with an aberrant expression in both HCC and other solid tumors, we must list: miR221/222 (up-regulated in HCC, colon, pancreas and gastric cancer); miR-21 (upregulated in HCC, ovarian, lung, breast cancer and glioblastoma); miR-199a, 200b and 214 (down-regulated in HCC and ovarian cancer) and miR-199b (down-regulated in HCC, ovarian and lung cancer). Importantly, genes frequently de-regulated in HCC, such as p27/CDKN1B

such as HPV [60].

angiogenesis [65, 66].

transcriptional regulation of target genes.

could be responsible for miRNA deregulation in tumors [63].

During the last years it has been shown a significant correspondence between clinical and humoral parameters (tumor size, differentiation grading, HBV or HCV infection, serum alfafeto protein (AFP)) and different molecular pathways activated in HCC cases: beta-catenin and Axin-1 mutated cases, with early relapse, behave in a different way comparedto cases with c-Myc and AKT activation associated with Interferon target genes inhibition, but also com‐ pared to hepatocellular carcinoma with overexpression of p53 and p21, the latter showing better differentiation and lower size [56]. Subclasses S1,S2,S3 defined on the basis of above mentioned clinical, humoraland morphological parametersare oftenassociated with different molecular profiles: S1 show Beta catenin and Axin mutation; S2 activation of c-Myc, AFP overexpression, AKT activation, IFN target genes inhibition; S3 cases are associated with better histological differentiation, overexpression of p53, p21, gene related with glycolipidic and alcohol metabolism, oxygen radical scavenging and coagulation [56]. The effort is the identi‐ fication of a classification system on the basis or with the contribution of the molecular changes of the tumors.

At this regard, miRNAs can play a crucial role. In fact, each of these small endogenous non-coding RNAs is characterized by the ability to transcriptionally or post-transcriptional‐ ly regulate many different target genes, thus being responsible of complex molecular changes [57].

In fact, these sequences favor mRNA degradation through sequence-specific interaction with the 3' untranslated region (3'-UTR) of targeted mRNAs; when the sequence is totally comple‐ mentary, miRNA pairing will induce mRNA degradation and are involved in development, apoptosis, proliferation and differentiation processes [58]. Alternatively, sequences partially complementary will more probably induce a stop in translation, without mRNA degradation. Therefore, a single miRNA is able to regulate the genic expression of hundreds of target genes.Its altered expression could cause a "post transcriptional collapse", i.e. the contemporary deregulation of multiple tumor suppressor or oncogenes whose sequences are complementary to the considered miRNA [59], thus deregulating many molecular pathways which could favor a malignant cellular phenotype [57]. In fact, the global effect of inactivation of a miRNA molecule will be the over-expression of its target genes, while its activation will induce the down-regulation of a lot of target genes. If deregulated miRNA target genes are involved in the regulation of important biological processes, such as apoptosis, cell cycle, tumoral cells invasiveness or angiogenesis, then the risk of uncontrolled growth and tumor development will increase [58].

Accordingly, in HCC has been described a deregulation of miRNA expression [57]. The fact that HCC-related changes in miRNA expression are absent in non-neoplastic parenchyma and the association of these changes with other neoplasms confirm the hypothesis of miRNA involvement in HCC pathogenesis.

Interestingly, more than 50% of miRNA genes are located in fragile chromosomal site or in cancer-associated genomic regions [60].

Many experimental evidences support the relationship between fragile sites and DNA instability in cancerous cells. In fact, fragile sites are preferential sites for chromatids exchang‐ es, translocations, deletions, gene amplification or integrations of associated viral sequences, such as HPV [60].

**3. Molecular changes in the development of HCC: The novel role of miRNA**

During the last years it has been shown a significant correspondence between clinical and humoral parameters (tumor size, differentiation grading, HBV or HCV infection, serum alfafeto protein (AFP)) and different molecular pathways activated in HCC cases: beta-catenin and Axin-1 mutated cases, with early relapse, behave in a different way comparedto cases with c-Myc and AKT activation associated with Interferon target genes inhibition, but also com‐ pared to hepatocellular carcinoma with overexpression of p53 and p21, the latter showing better differentiation and lower size [56]. Subclasses S1,S2,S3 defined on the basis of above mentioned clinical, humoraland morphological parametersare oftenassociated with different molecular profiles: S1 show Beta catenin and Axin mutation; S2 activation of c-Myc, AFP overexpression, AKT activation, IFN target genes inhibition; S3 cases are associated with better histological differentiation, overexpression of p53, p21, gene related with glycolipidic and alcohol metabolism, oxygen radical scavenging and coagulation [56]. The effort is the identi‐ fication of a classification system on the basis or with the contribution of the molecular changes

At this regard, miRNAs can play a crucial role. In fact, each of these small endogenous non-coding RNAs is characterized by the ability to transcriptionally or post-transcriptional‐ ly regulate many different target genes, thus being responsible of complex molecular

In fact, these sequences favor mRNA degradation through sequence-specific interaction with the 3' untranslated region (3'-UTR) of targeted mRNAs; when the sequence is totally comple‐ mentary, miRNA pairing will induce mRNA degradation and are involved in development, apoptosis, proliferation and differentiation processes [58]. Alternatively, sequences partially complementary will more probably induce a stop in translation, without mRNA degradation. Therefore, a single miRNA is able to regulate the genic expression of hundreds of target genes.Its altered expression could cause a "post transcriptional collapse", i.e. the contemporary deregulation of multiple tumor suppressor or oncogenes whose sequences are complementary to the considered miRNA [59], thus deregulating many molecular pathways which could favor a malignant cellular phenotype [57]. In fact, the global effect of inactivation of a miRNA molecule will be the over-expression of its target genes, while its activation will induce the down-regulation of a lot of target genes. If deregulated miRNA target genes are involved in the regulation of important biological processes, such as apoptosis, cell cycle, tumoral cells invasiveness or angiogenesis, then the risk of uncontrolled growth and tumor development

Accordingly, in HCC has been described a deregulation of miRNA expression [57]. The fact that HCC-related changes in miRNA expression are absent in non-neoplastic parenchyma and the association of these changes with other neoplasms confirm the hypothesis of miRNA

Interestingly, more than 50% of miRNA genes are located in fragile chromosomal site or in

of the tumors.

182 Hepatocellular Carcinoma - Future Outlook

changes [57].

will increase [58].

involvement in HCC pathogenesis.

cancer-associated genomic regions [60].

Besides the association with fragile sites, microRNA genes may be involved in tumorigenesis process through other mechanisms, such as point mutation, deletion, amplification, translo‐ cation or epigenetic modifications [61]. Most known microRNA have been identified within genomic cancer-associated regions: minimal regions of loss of heterozigosity, where often oncosuppressor genes are present or minimal region of amplification, where often oncogenes have been identified [62]. This strongly suggest that miRNAgenes may behave either as oncogenes or as tumor suppressor genes and, in particular, the same miRNA may behave in different way on the basis of the kind of alteration, cellular type or transcriptional/posttranscriptional regulation of target genes.

miRNA deregulation can also be the consequence of epigenetic changes, In fact, an extensive genomic analysis of miRNA-coding gene sequences showed that about 50% of these loci are CpG island associated [62] makingpossible that an alteration in DNA methylation/acethylation could be responsible for miRNA deregulation in tumors [63].

Regarding the changes in miRNA expression in different tumors, the Volinia study [64], performed a microarray analysis of 20 different miRNAs in 540 specimen obtained from different tumor types (lung, breast, stomach, prostate, colon, pancreas) identifying a miRNA signature common to some different solid tumors). The value of this study, find a confirma‐ tion in the similar grouping, between different tumors, of the miRNA expression signa‐ ture: this suggest a common mechanism of involvement of miRNA in human carcinogenesis. Prostate, colon, stomach, pancreas show mostly similar signature among them, whereas lung and breast are represented by a fairly different signature. Furthermore, within the signature, some miRNA have been found whose association with other human tumors was already known: among them there are miR-17-5p, miR-20a, miR-21, miR-92, miR-106a, e miR-155, and molecular targets of these miRNA are significantly rich in tumor suppres‐ sor genes and oncogenes. These data suggest the crucial involvement of miRNA in the pathogenesis of solid tumors and confirm the function of miRNA as dominant and recessive tumoral genes. Furthermore, it has been shown the role of miRNA not only in the early phases of primitive tumors development, but also during the progression and the metastat‐ ic diffusion of the neoplastic disease. In fact, many experimental evidences show miRNA involvement in the regulation of biological process leading to the acquisition of metastat‐ ic potential, such as adhesion, invasion, migration, epithelial-to-mesenchymal transition and angiogenesis [65, 66].

Among miRNAs with an aberrant expression in both HCC and other solid tumors, we must list: miR221/222 (up-regulated in HCC, colon, pancreas and gastric cancer); miR-21 (upregulated in HCC, ovarian, lung, breast cancer and glioblastoma); miR-199a, 200b and 214 (down-regulated in HCC and ovarian cancer) and miR-199b (down-regulated in HCC, ovarian and lung cancer). Importantly, genes frequently de-regulated in HCC, such as p27/CDKN1B and p57/CDKN1C or PTEN, have been identified as targets of miR-221/222 and miR-21 targets (see below).

It is worth noting that another mi-RNA under-expressed in most HCC [75], miR-199a-3p, is capable of regulating c-Met oncogene expression [76]:these data suggest that various deregu‐ lated miRNA in a specific neoplasia may modulate the same target gene, leading to strong alteration of the molecular pathways downstream the target gene, thus influencing the tumor

Conditions that Predispose to the Development of HCC: The Role of Tumor Associated Fibroblasts and of microRNA

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185

RAS oncogene overexpression is associated to miRNA deregulation without oncogene point mutation. Pathway alteration can be induced by downregulation of let-7 miRNA family

Regarding the biological processes controlled by miRNAs, they play a direct role on cellular growth control. As previously mentioned, miR-221 is up-regulated in many neoplasms and influence the expression of the cyclin-dependent kinase inhibitor CDKN1B/p27, which controls the cell cycle progression [57]. The activation of pathways involving PI3K/AKT leads to phosphorylation of proteins that favor cell survival, under the control of tumor suppressor lipid phosphatase PTEN, which is a direct miR-21 target; in vitro studies showed the associa‐ tion between mir-21 inhibition and PTEN overexpression, with subsequent decreased neo‐ plastic cell proliferation and invasion [57]. Moreover, MiR-21 inhibition induces changes in metalloproteinases 2 and 9 expression: both these molecules are downstream PTEN mediators with a known role in cellular migration and invasion [57]. These data suggest that the aberrant miR-21 expression may favor HCC growth and diffusion through the modulation of the expression of PTEN and of PTEN-dependent pathways involved in neoplastic cells phenotype and behavior modulation. Again, cyclin G1 upregulation following miR-122 down-regulation

Regarding the role of miRNA as biomarkers, there is evidence that, in HCC, miRNAs could

As already underlined, some known molecular factors characterizing HCC may play a role as prognostic factors (for example c-MET or p27); similarly, there is evidence that some deregu‐ lated miR [58] may allow, in association with clinical parameters and histological patterns, classifying HCC, thus identifying new criteria for prognostic stratification [57]. This is also possible due to distinct miRNA profiles of different hepatocellular carcinoma subtypes (at variance with similar signature between tumors in different organs)Such a differentiation takes place on the basis of supposed mechanism of origin (cancer stem cells or mature hepatocytelike): the different groups show distinctbiological behavior. Furthermore,HBV or HCV etiology of basic disease and primary or metastatic nature of the neoplastic lesion are associated

Regarding the potential role of miRNAs as diagnostic biomarkers, at least 20 miRNAs show different changes between lesions with different origin; cancer stem cells origin is associated with self-renewal capability, differentiation and aggressive tumorigenesis in vivo: miR-181 family members, over-expressed in HCC, favor the origin of neoplasia from progenitor cells through an influence on CDX2 and GATA6, differentiation related genes, and on NLK a WNT/

may induce p53 down-regulation, so favoring tumorigenesis [57].

have a prognostic as well as a diagnostic role.

with different miRNA signatures in HCC [60].

Beta catenin pathway inhibitor.

progression.

component.

Considering HCC-specific miRNAs, miR-122 is one of the best characterized molecules. miR-122 is, in fact, a liver specific miRNA, representing 70% of all liver expressed miRNAs, and it is down-regulated in most human and murine HCC. Its deregulation induces important changes in the phenotype of adult hepatocytes and in several liver functions, such as lipid metabolism [67] and cholesterol synthesis [68]. These data allow hypothesizing a correlation between the lowered miR-122 expression in HCC and the loss of hepatic differentiation in neoplastic cells. Furthermore, Jopling*et al*have demonstrated that miR-122 is able to bind to 5'- UTR RNA region of HCV [69]: this region is preserved in all six viral genotypes, so suggesting that miR-122 is an essential element for virus replication in hepatocytes. It has been shown that functional inactivation of miR-122 induces the 80% reduction of viral replication, suggesting that miR-122 inactivation in hepatocellular carcinoma could increase neoplastic cells resistance to HCV replication.

Concerning the miRNA involvement in different steps of hepatocellular carcinoma progres‐ sion, it has been demonstrated a correlation between miR-222, miR-106, miR-92, miR-17-5p, miR-20, miR-18 and hepatocellular carcinoma differentiation grading, suggesting the involve‐ ment of a restricted number of miRNA in neoplastic progression [70]. According to this data a 20 miRNAs signature has been reported [71], capable to distinguish primitive hepatocellular carcinoma metastasizing through venous channels from solitary, non-metastatic tumors. Furthermore, a predictive analysis revealed that most of 20 miRNA of HCC signature are associated with patients' survival. These signatures could then represent a simple method for a diagnostic/prognostic profiling, capable of identifying HCC patients at high risk of devel‐ oping a metastatic disease or a liver relapse.

Other authors evaluate the predictive accuracy in distinguishing neoplastic and non-neoplastic parenchyma equal to97,8% on the basis of 8 miRNAs, while others again identify 18 overex‐ pressed miRNA in hepatocellular carcinoma and only 6 overexpressedin non-neoplastic tissue [72] (and with aberrant miR-21 expression associated with metastasis risk and connected with PTEN targeting; miR-224 could act on genes apoptosis regulating (API-5) [73, 74].

Another miRNA involved in tumor progression and diffusion is miR-34a, a transcriptional target of p53, deleted in many human neoplastic lesions. Besides molecular targets involved in cell cycle progression, miR-34a regulates the expression of the oncoprotein c-Met, a tyrosin kinase receptor activated by the hepatocyte growth factor binding, able to induce the phos‐ phorilation of molecules responsible for signal transduction, such as ERK1/2, thus exerting the function of key factors in tumor invasion and migration regulation.

A recent study [66] reported a significant reduction of miR-34a expression in most carcinom‐ atous liver tissue with respect to non-neoplastic adjacent liver tissue, with an inverse correla‐ tion between miR-34a-and c-Met-expression levels. Furthermore, low levels of miR-34a positively relate with the development of metastatic disease and neoplastic vascular invasion. Accordingly, in vitro studies showed a lower migration and metastatic potential of neoplastic cells after miR-34a c-Met dependent suppression.

It is worth noting that another mi-RNA under-expressed in most HCC [75], miR-199a-3p, is capable of regulating c-Met oncogene expression [76]:these data suggest that various deregu‐ lated miRNA in a specific neoplasia may modulate the same target gene, leading to strong alteration of the molecular pathways downstream the target gene, thus influencing the tumor progression.

and p57/CDKN1C or PTEN, have been identified as targets of miR-221/222 and miR-21 targets

Considering HCC-specific miRNAs, miR-122 is one of the best characterized molecules. miR-122 is, in fact, a liver specific miRNA, representing 70% of all liver expressed miRNAs, and it is down-regulated in most human and murine HCC. Its deregulation induces important changes in the phenotype of adult hepatocytes and in several liver functions, such as lipid metabolism [67] and cholesterol synthesis [68]. These data allow hypothesizing a correlation between the lowered miR-122 expression in HCC and the loss of hepatic differentiation in neoplastic cells. Furthermore, Jopling*et al*have demonstrated that miR-122 is able to bind to 5'- UTR RNA region of HCV [69]: this region is preserved in all six viral genotypes, so suggesting that miR-122 is an essential element for virus replication in hepatocytes. It has been shown that functional inactivation of miR-122 induces the 80% reduction of viral replication, suggesting that miR-122 inactivation in hepatocellular carcinoma could increase neoplastic cells resistance

Concerning the miRNA involvement in different steps of hepatocellular carcinoma progres‐ sion, it has been demonstrated a correlation between miR-222, miR-106, miR-92, miR-17-5p, miR-20, miR-18 and hepatocellular carcinoma differentiation grading, suggesting the involve‐ ment of a restricted number of miRNA in neoplastic progression [70]. According to this data a 20 miRNAs signature has been reported [71], capable to distinguish primitive hepatocellular carcinoma metastasizing through venous channels from solitary, non-metastatic tumors. Furthermore, a predictive analysis revealed that most of 20 miRNA of HCC signature are associated with patients' survival. These signatures could then represent a simple method for a diagnostic/prognostic profiling, capable of identifying HCC patients at high risk of devel‐

Other authors evaluate the predictive accuracy in distinguishing neoplastic and non-neoplastic parenchyma equal to97,8% on the basis of 8 miRNAs, while others again identify 18 overex‐ pressed miRNA in hepatocellular carcinoma and only 6 overexpressedin non-neoplastic tissue [72] (and with aberrant miR-21 expression associated with metastasis risk and connected with

Another miRNA involved in tumor progression and diffusion is miR-34a, a transcriptional target of p53, deleted in many human neoplastic lesions. Besides molecular targets involved in cell cycle progression, miR-34a regulates the expression of the oncoprotein c-Met, a tyrosin kinase receptor activated by the hepatocyte growth factor binding, able to induce the phos‐ phorilation of molecules responsible for signal transduction, such as ERK1/2, thus exerting the

A recent study [66] reported a significant reduction of miR-34a expression in most carcinom‐ atous liver tissue with respect to non-neoplastic adjacent liver tissue, with an inverse correla‐ tion between miR-34a-and c-Met-expression levels. Furthermore, low levels of miR-34a positively relate with the development of metastatic disease and neoplastic vascular invasion. Accordingly, in vitro studies showed a lower migration and metastatic potential of neoplastic

PTEN targeting; miR-224 could act on genes apoptosis regulating (API-5) [73, 74].

function of key factors in tumor invasion and migration regulation.

cells after miR-34a c-Met dependent suppression.

(see below).

184 Hepatocellular Carcinoma - Future Outlook

to HCV replication.

oping a metastatic disease or a liver relapse.

RAS oncogene overexpression is associated to miRNA deregulation without oncogene point mutation. Pathway alteration can be induced by downregulation of let-7 miRNA family component.

Regarding the biological processes controlled by miRNAs, they play a direct role on cellular growth control. As previously mentioned, miR-221 is up-regulated in many neoplasms and influence the expression of the cyclin-dependent kinase inhibitor CDKN1B/p27, which controls the cell cycle progression [57]. The activation of pathways involving PI3K/AKT leads to phosphorylation of proteins that favor cell survival, under the control of tumor suppressor lipid phosphatase PTEN, which is a direct miR-21 target; in vitro studies showed the associa‐ tion between mir-21 inhibition and PTEN overexpression, with subsequent decreased neo‐ plastic cell proliferation and invasion [57]. Moreover, MiR-21 inhibition induces changes in metalloproteinases 2 and 9 expression: both these molecules are downstream PTEN mediators with a known role in cellular migration and invasion [57]. These data suggest that the aberrant miR-21 expression may favor HCC growth and diffusion through the modulation of the expression of PTEN and of PTEN-dependent pathways involved in neoplastic cells phenotype and behavior modulation. Again, cyclin G1 upregulation following miR-122 down-regulation may induce p53 down-regulation, so favoring tumorigenesis [57].

Regarding the role of miRNA as biomarkers, there is evidence that, in HCC, miRNAs could have a prognostic as well as a diagnostic role.

As already underlined, some known molecular factors characterizing HCC may play a role as prognostic factors (for example c-MET or p27); similarly, there is evidence that some deregu‐ lated miR [58] may allow, in association with clinical parameters and histological patterns, classifying HCC, thus identifying new criteria for prognostic stratification [57]. This is also possible due to distinct miRNA profiles of different hepatocellular carcinoma subtypes (at variance with similar signature between tumors in different organs)Such a differentiation takes place on the basis of supposed mechanism of origin (cancer stem cells or mature hepatocytelike): the different groups show distinctbiological behavior. Furthermore,HBV or HCV etiology of basic disease and primary or metastatic nature of the neoplastic lesion are associated with different miRNA signatures in HCC [60].

Regarding the potential role of miRNAs as diagnostic biomarkers, at least 20 miRNAs show different changes between lesions with different origin; cancer stem cells origin is associated with self-renewal capability, differentiation and aggressive tumorigenesis in vivo: miR-181 family members, over-expressed in HCC, favor the origin of neoplasia from progenitor cells through an influence on CDX2 and GATA6, differentiation related genes, and on NLK a WNT/ Beta catenin pathway inhibitor.

MiR-21, miR-10b, miR-222 and miR-224 are highly represented in HCC, while non-malignant hepatic lesions show decreased miR-202 and miR-203 expression. Alcohol consumption related hepatocellular carcinoma shows low miR-126 levels, while high levels of miR-96 are present in neoplastic lesions HBV associated: such alterations have not been shown in nonneoplastic parenchyma [58]. Furthermore, hepatocellular carcinoma cases with high risk of metastatic diffusion and cases without metastasis risk present distinct miRNA groups' expression.

hypothesis that liver resident MASCs could generate TAFs in pathological conditions: a population of MASCs with TAFs characteristics both in human hepatocellular carcinoma and cirrhotic liver has been demonstrated, so suggesting a possible role in the development of neoplasia in the adequate environment [2]. On the other hand, TAFs from non-neoplastic and non-cirrhotic livers do not show aberrant growth properties: so, TAFs can originate from

Conditions that Predispose to the Development of HCC: The Role of Tumor Associated Fibroblasts and of microRNA

http://dx.doi.org/10.5772/56951

187

The in vivo counterpart of MASCs is still undefined, but the interesting data concern their sharing of some feature with activated hepatic stellate cells [85-87]. This aspect is of particular interest, given the hypothesis that stellate cells could be progenitor cells: in fact, the expression of OCT-4 and of markers of all the three germ layers and their ability to give rise in vitro to

A fate-mapping study showed that stellate cells could become oval cells when activated in liver injury, and that these cells participate in ductular proliferation [88]. This offers a sub‐ stantial contribution to the discussed sharing of pathways and cells between developmental,

All these evidences point to the presence of mesenchymal, widely multipotent cells in adult tissues,whichcantakeparttoregenerativeaswellasinflammatoryandneoplasticprocesses[89].

In the paper by Cesselli et al 2011 [2] it is well documented that MASCs isolated from hepa‐ tocellular carcinoma have the main characteristic of TAFs, at variance with cells from nonneoplastic and non-cirrhotic livers. TAFs act on the tumor growth in different ways, which are able to modify the microenvironment in the sense of a more suitable situation for the increase of tumor growth [48, 78, 90]. TAFs are contractile cells, with a strict spatial relation with blood vessels; they produce growth factors (HGF, TGF beta, EGF, bFGF, IGF), cytokines, chemokines, enzymes; they can degrade the extracellular matrix and can behave as immunomodulating cells [48, 78, 90]. Finally, increased metastatic potential and poor prognosis are associated with

Tumor cell lines medium can induce in L-MASCs aberrant growth properties and the ability to produce specific TAF markers [47, 48, 78]. The origin of TAFs seems to be connected with four possible sources: epithelial-mesenchymal transition of the neoplastic cells (associated with genetic changes within the TAFs); recruitment and activation of resident fibroblasts; recruitment of circulating/bone marrow derived mesenchymal stem cells; recruitment of mesenchymal stem cells [48, 78, 82]. In the paper from Cesselli, Beltrami et al. it has been shown the origin of TAFs from a population of resident primitive cells with mesenchymal features [2].The Authors also underline that blocking the activation of TAFs and their continuous communication with the cancer cells could, in conjunction with chemotherapy regimens, limit tumor progression and metastasis [45, 82, 91]: the much lower instability of TAFs versus malignant cells seems to make them less likely to undergo the onset of resistance to chemo‐

endothelial cells and hepatocytes offer essential elements [2].

regenerative and neoplastic processes in the liver.

resident primitive cells [2].

the presence of TAFs [48].

therapy drugs [81, 92].

Low miR-375 expression have been shown either in hepatocellular adenoma or in carcinoma with Beta catenin mutation: the significant inverse correlation between miR-375 level and Beta catenin targeted gene expression suggests a direct relationship between Beta catenin activation and miR-375 repression [58]. Recently a role of survival predictor and IFN adjuvant therapy response in hepatocellular carcinoma for miR-26 has been identified [77].

Finally, as previously mentioned, genetic mutations and transcriptional, epigenetic changes may induce miRNA alterations. In the case of HCC it has been shown the role of endoribonu‐ clease III DICER for miRNA maturation: its role in cleavage and maturation of miRNA renders impossible for the cell a full expression of miRNA if endoribonuclease is disrupted. DICER deficient hepatocytes loose the expression of all miRNA, with expression of liver specific fetal genes and deregulation of much genes related to neoplastic development [60].

In conclusion, up to date experimental evidences show that hepatocellular carcinoma subtypes are characterized by distinct miRNA expression profiles, related to grade of aggressiveness, risk factors and genetic changes. MiRNAs may therefore represent not only useful diagnostic and prognostic markers, but important target molecules for potential therapeutic treatment.

### **4. Tumor associated fibroblast in the development of HCC**

If molecular changes take place in liver parenchymal cells undergoing to inflammatory and fibrotic parenchymal damage, leading to the phenomena that characterize tumoral initiation and progression, then, it is also an important matter the evaluation of the role of inflammatory conditions and fibrosis in stromal changes, and, particularly, in tumor associated fibroblasts development.

Among the constituents of the tumor microenvironment, tumor associated fibroblasts (TAF) play a key role in tumor progression, angiogenesis, growth and metastasis: they are charac‐ terized by the expression of specific markers [45, 78-82] and seems to assume a role in clinical tumor prognosis.

Recently [83, 84] it has been optimized a method to isolate, from several human adult tissues, a population of primitive cells, named multipotent adult stem cells (MASCs) with mesenchy‐ malimmunophenotype, clonogenicity and multiple in vitro differentiation capacity [83, 84]. MASCs isolated both from neoplastic and from cirrhotic human liver tissue allow to test the hypothesis that liver resident MASCs could generate TAFs in pathological conditions: a population of MASCs with TAFs characteristics both in human hepatocellular carcinoma and cirrhotic liver has been demonstrated, so suggesting a possible role in the development of neoplasia in the adequate environment [2]. On the other hand, TAFs from non-neoplastic and non-cirrhotic livers do not show aberrant growth properties: so, TAFs can originate from resident primitive cells [2].

MiR-21, miR-10b, miR-222 and miR-224 are highly represented in HCC, while non-malignant hepatic lesions show decreased miR-202 and miR-203 expression. Alcohol consumption related hepatocellular carcinoma shows low miR-126 levels, while high levels of miR-96 are present in neoplastic lesions HBV associated: such alterations have not been shown in nonneoplastic parenchyma [58]. Furthermore, hepatocellular carcinoma cases with high risk of metastatic diffusion and cases without metastasis risk present distinct miRNA groups'

Low miR-375 expression have been shown either in hepatocellular adenoma or in carcinoma with Beta catenin mutation: the significant inverse correlation between miR-375 level and Beta catenin targeted gene expression suggests a direct relationship between Beta catenin activation and miR-375 repression [58]. Recently a role of survival predictor and IFN adjuvant therapy

Finally, as previously mentioned, genetic mutations and transcriptional, epigenetic changes may induce miRNA alterations. In the case of HCC it has been shown the role of endoribonu‐ clease III DICER for miRNA maturation: its role in cleavage and maturation of miRNA renders impossible for the cell a full expression of miRNA if endoribonuclease is disrupted. DICER deficient hepatocytes loose the expression of all miRNA, with expression of liver specific fetal

In conclusion, up to date experimental evidences show that hepatocellular carcinoma subtypes are characterized by distinct miRNA expression profiles, related to grade of aggressiveness, risk factors and genetic changes. MiRNAs may therefore represent not only useful diagnostic and prognostic markers, but important target molecules for potential

If molecular changes take place in liver parenchymal cells undergoing to inflammatory and fibrotic parenchymal damage, leading to the phenomena that characterize tumoral initiation and progression, then, it is also an important matter the evaluation of the role of inflammatory conditions and fibrosis in stromal changes, and, particularly, in tumor associated fibroblasts

Among the constituents of the tumor microenvironment, tumor associated fibroblasts (TAF) play a key role in tumor progression, angiogenesis, growth and metastasis: they are charac‐ terized by the expression of specific markers [45, 78-82] and seems to assume a role in clinical

Recently [83, 84] it has been optimized a method to isolate, from several human adult tissues, a population of primitive cells, named multipotent adult stem cells (MASCs) with mesenchy‐ malimmunophenotype, clonogenicity and multiple in vitro differentiation capacity [83, 84]. MASCs isolated both from neoplastic and from cirrhotic human liver tissue allow to test the

response in hepatocellular carcinoma for miR-26 has been identified [77].

genes and deregulation of much genes related to neoplastic development [60].

**4. Tumor associated fibroblast in the development of HCC**

expression.

186 Hepatocellular Carcinoma - Future Outlook

therapeutic treatment.

development.

tumor prognosis.

The in vivo counterpart of MASCs is still undefined, but the interesting data concern their sharing of some feature with activated hepatic stellate cells [85-87]. This aspect is of particular interest, given the hypothesis that stellate cells could be progenitor cells: in fact, the expression of OCT-4 and of markers of all the three germ layers and their ability to give rise in vitro to endothelial cells and hepatocytes offer essential elements [2].

A fate-mapping study showed that stellate cells could become oval cells when activated in liver injury, and that these cells participate in ductular proliferation [88]. This offers a sub‐ stantial contribution to the discussed sharing of pathways and cells between developmental, regenerative and neoplastic processes in the liver.

All these evidences point to the presence of mesenchymal, widely multipotent cells in adult tissues,whichcantakeparttoregenerativeaswellasinflammatoryandneoplasticprocesses[89].

In the paper by Cesselli et al 2011 [2] it is well documented that MASCs isolated from hepa‐ tocellular carcinoma have the main characteristic of TAFs, at variance with cells from nonneoplastic and non-cirrhotic livers. TAFs act on the tumor growth in different ways, which are able to modify the microenvironment in the sense of a more suitable situation for the increase of tumor growth [48, 78, 90]. TAFs are contractile cells, with a strict spatial relation with blood vessels; they produce growth factors (HGF, TGF beta, EGF, bFGF, IGF), cytokines, chemokines, enzymes; they can degrade the extracellular matrix and can behave as immunomodulating cells [48, 78, 90]. Finally, increased metastatic potential and poor prognosis are associated with the presence of TAFs [48].

Tumor cell lines medium can induce in L-MASCs aberrant growth properties and the ability to produce specific TAF markers [47, 48, 78]. The origin of TAFs seems to be connected with four possible sources: epithelial-mesenchymal transition of the neoplastic cells (associated with genetic changes within the TAFs); recruitment and activation of resident fibroblasts; recruitment of circulating/bone marrow derived mesenchymal stem cells; recruitment of mesenchymal stem cells [48, 78, 82]. In the paper from Cesselli, Beltrami et al. it has been shown the origin of TAFs from a population of resident primitive cells with mesenchymal features [2].The Authors also underline that blocking the activation of TAFs and their continuous communication with the cancer cells could, in conjunction with chemotherapy regimens, limit tumor progression and metastasis [45, 82, 91]: the much lower instability of TAFs versus malignant cells seems to make them less likely to undergo the onset of resistance to chemo‐ therapy drugs [81, 92].

#### **5. Conclusions**

Liver parenchyma, exposed to long standing viral or toxic damage, undergoes a diffuse, fibrosing rearrangement, developing cirrhosis. In this context, genetic changes in the cellular component, the activation of different pathways, the production of cytokines and growth factors, phenotypic changes of epithelial or mesenchymal cells produce complex phenomena, involving regenerative, preneoplastic and neoplastic changes.The role of liver cells and/or progenitor cells in neoplastic growth is heavily influenced by mesenchymal component, in particular by TAFs. Cirrhotic livers, when not yet neoplastic, already possess a population of multipotent adult stem cells with TAFs properties.

**Author details**

S. Uzzau3

Udine, Italy

**References**

Italy

C. Avellini1\*, D. Cesselli2

, A.P. Beltrami2

\*Address all correspondence to: avellini.claudio@aoud.sanita.fvg.it

cinoma. Nat Genet. 2002 Aug;31(4):339-46.

[3] Mcsween. Pathology of the liver. Livingstone C, editor2002.

good, the bad and the ugly. J Pathol. 2009 Jan;217(2):282-98.

trointest Liver Physiol. 2011 Sep;301(3):G425-34.

nogenesis. J Biomed Biotechnol. 2010;2010:984248.

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Hepatol. 2011;2011:120925.

Nov;41(5):864-80.

2010;2010:272170.

, M. Orsaria1

Conditions that Predispose to the Development of HCC: The Role of Tumor Associated Fibroblasts and of microRNA

1 Department of Pathology, University Hospital "S. Maria dellaMisericordia" of Udine,

3 Department of Surgery, University Hospital "S. Maria dellaMisericordia" of Udine, Udine,

[1] Thorgeirsson SS, Grisham JW. Molecular pathogenesis of human hepatocellular car‐

[2] Cesselli D, Beltrami AP, Poz A, Marzinotto S, Comisso E, Bergamin N, et al. Role of tumor associated fibroblasts in human liver regeneration, cirrhosis, and cancer. Int J

[4] Patsenker E, Stickel F. Role of integrins in fibrosing liver diseases. Am J PhysiolGas‐

[5] Alison MR, Islam S, Lim S. Stem cells in liver regeneration, fibrosis and cancer: the

[6] Semela D, Dufour JF. Angiogenesis and hepatocellular carcinoma. J Hepatol. 2004

[7] Sanz-Cameno P, Trapero-Marugan M, Chaparro M, Jones EA, Moreno-Otero R. An‐ giogenesis: from chronic liver inflammation to hepatocellular carcinoma. J Oncol.

[8] Kung JW, Currie IS, Forbes SJ, Ross JA. Liver development, regeneration, and carci‐

[9] Riehle KJ, Dan YY, Campbell JS, Fausto N. New concepts in liver regeneration. J Gas‐

2 Department of Medical and Biological Sciences, University of Udine, Udine, Italy

, S. Marzinotto1

, F. Morassi1

and

http://dx.doi.org/10.5772/56951

189

The possible origin of TAFs from a population of primitive mesenchymal stem cells gives to the tumor supporting stroma a more complex and important role. In fact, from " rare resident stem cells, such as multipotent adult stem cells (MASCs) " the entire formation and instruction of the elements of supportive microenvironment could take place [2]. MASCs differentiation potential " into many stromal cell types and their molecular signature consisting of overex‐ pression " of a gene panel involved in extracellular matrix remodeling, immunomodulation, and cytokines and growth factors production [83] offer a key to understand the relationship between liver parenchyma, neoplastic growth and stromal component.

" Multipotent adult stem cells isolated from healthy livers can acquire a TAF phenotype when grown in conditional medium from tumor cell lines, suggesting that multipotent cells residing in the liver may represent a population " able to stroma formation and contributing to tumor progression, in adequate conditions [2]. The effort to identify novel therapies aimed at interfering with the interaction between stroma and cancer cells represent a crucial perspective.

The discovery, in 1993, by Ambros and Ruvkun of this new mechanism of gene regulation, with large mRNA under the control of a small RNA opened a new way to develop the understanding of the regulation of critical cellular process (cell division, metabolism, devel‐ opment and death) by micro RNA. MiRNA can target more than 500 mRNA and a single mRNA can be targeted by multiple miRNA. The specificity of targets depend on the degree of base complementarities to the target mRNA: when the miRNA have perfect base comple‐ mentarities, than messenger RNA undergo degradation; if there is not perfect complementar‐ ity, than posttranslational inhibition occur. Through this mechanism, miRNA can exert an essential control on a lot of processes, in particular in neoplasia harboring and diffusion, opening a crucial field in prognostic prediction and in possible intervention to break the carcinogenetic process and to obtain the disease control through target therapy.

The concepts of multidirectional differentiation of mesenchymal stem cells, of tumor associ‐ ated fibroblasts, of cancerization fields, epithelial-mesenchymal transition and mesenchymalepithelial transition, miRNA deregulation,the progress in the identification of reproducible classification criteria on the basis of hepatocellular carcinomamolecular changes offer new insights concerning liver neoplastic growths, with key points concerning the association between developmental, regenerative and neoplastic growth mechanisms and the relationship between parenchymal hepatocytes and stromal component in preneoplastic and neoplastic liver diseases as the main ways for a better understanding of neoplastic liver biology.

### **Author details**

**5. Conclusions**

188 Hepatocellular Carcinoma - Future Outlook

multipotent adult stem cells with TAFs properties.

Liver parenchyma, exposed to long standing viral or toxic damage, undergoes a diffuse, fibrosing rearrangement, developing cirrhosis. In this context, genetic changes in the cellular component, the activation of different pathways, the production of cytokines and growth factors, phenotypic changes of epithelial or mesenchymal cells produce complex phenomena, involving regenerative, preneoplastic and neoplastic changes.The role of liver cells and/or progenitor cells in neoplastic growth is heavily influenced by mesenchymal component, in particular by TAFs. Cirrhotic livers, when not yet neoplastic, already possess a population of

The possible origin of TAFs from a population of primitive mesenchymal stem cells gives to the tumor supporting stroma a more complex and important role. In fact, from " rare resident stem cells, such as multipotent adult stem cells (MASCs) " the entire formation and instruction of the elements of supportive microenvironment could take place [2]. MASCs differentiation potential " into many stromal cell types and their molecular signature consisting of overex‐ pression " of a gene panel involved in extracellular matrix remodeling, immunomodulation, and cytokines and growth factors production [83] offer a key to understand the relationship

" Multipotent adult stem cells isolated from healthy livers can acquire a TAF phenotype when grown in conditional medium from tumor cell lines, suggesting that multipotent cells residing in the liver may represent a population " able to stroma formation and contributing to tumor progression, in adequate conditions [2]. The effort to identify novel therapies aimed at interfering with the interaction between stroma and cancer cells represent a crucial perspective. The discovery, in 1993, by Ambros and Ruvkun of this new mechanism of gene regulation, with large mRNA under the control of a small RNA opened a new way to develop the understanding of the regulation of critical cellular process (cell division, metabolism, devel‐ opment and death) by micro RNA. MiRNA can target more than 500 mRNA and a single mRNA can be targeted by multiple miRNA. The specificity of targets depend on the degree of base complementarities to the target mRNA: when the miRNA have perfect base comple‐ mentarities, than messenger RNA undergo degradation; if there is not perfect complementar‐ ity, than posttranslational inhibition occur. Through this mechanism, miRNA can exert an essential control on a lot of processes, in particular in neoplasia harboring and diffusion, opening a crucial field in prognostic prediction and in possible intervention to break the

between liver parenchyma, neoplastic growth and stromal component.

carcinogenetic process and to obtain the disease control through target therapy.

liver diseases as the main ways for a better understanding of neoplastic liver biology.

The concepts of multidirectional differentiation of mesenchymal stem cells, of tumor associ‐ ated fibroblasts, of cancerization fields, epithelial-mesenchymal transition and mesenchymalepithelial transition, miRNA deregulation,the progress in the identification of reproducible classification criteria on the basis of hepatocellular carcinomamolecular changes offer new insights concerning liver neoplastic growths, with key points concerning the association between developmental, regenerative and neoplastic growth mechanisms and the relationship between parenchymal hepatocytes and stromal component in preneoplastic and neoplastic

C. Avellini1\*, D. Cesselli2 , A.P. Beltrami2 , M. Orsaria1 , S. Marzinotto1 , F. Morassi1 and S. Uzzau3

\*Address all correspondence to: avellini.claudio@aoud.sanita.fvg.it

1 Department of Pathology, University Hospital "S. Maria dellaMisericordia" of Udine, Udine, Italy

2 Department of Medical and Biological Sciences, University of Udine, Udine, Italy

3 Department of Surgery, University Hospital "S. Maria dellaMisericordia" of Udine, Udine, Italy

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**Chapter 10**

**Signs and Symptoms**

http://dx.doi.org/10.5772/56162

resection, or liver transplantation.

**1. Introduction**

Valerio Barghini, Debora Donnini,

Alessandro Uzzau and Giorgio Soardo

Additional information is available at the end of the chapter

Hepatocellular carcinoma (HCC) is often diagnosed after the tumor manifests clinical signs and symptoms. Early diagnosis is usually performed thanks to HCC screening programs for patients affected by liver cirrhosis or chronic viral hepatopathies using ultrasound and serum alfa-fetoprotien. In most HCC cases, clinical signs and symptoms of this tumor may occur several months after development, when therapy can not be curative, given the advanced tumor stage and underlying liver disease, which preclude curative options, such as ablation,

Clinical features of HCC are often similar to those caused by the underlying hepatic disease. It is very hard for physicians to distinguish signs and symptoms of HCC in contests charac‐ terized by an advanced liver disease. Advanced liver cancer can be responsible for accelerated

In this chapter, we review the clinical signs and symptoms induced by advanced carcinoma. We also discuss particular clinical scenarios caused by metastases and paraneoplastic syn‐

Non-specific systemic signs and symptoms as asthenia, anorexia, weight loss, and nausea, are often present in patients with HCC (table 1). HCC should be suspected with the onset of these

> © 2013 Barghini et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

liver functions deterioration caused by the intrahepatic tumor growth.

dromes, sometimes described case reports in literature.

**2. Non-specific signs and symptoms**

clinical features in patients at risk for this tumor.

**Chapter 10**

## **Signs and Symptoms**

Valerio Barghini, Debora Donnini, Alessandro Uzzau and Giorgio Soardo

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/56162

**1. Introduction**

Hepatocellular carcinoma (HCC) is often diagnosed after the tumor manifests clinical signs and symptoms. Early diagnosis is usually performed thanks to HCC screening programs for patients affected by liver cirrhosis or chronic viral hepatopathies using ultrasound and serum alfa-fetoprotien. In most HCC cases, clinical signs and symptoms of this tumor may occur several months after development, when therapy can not be curative, given the advanced tumor stage and underlying liver disease, which preclude curative options, such as ablation, resection, or liver transplantation.

Clinical features of HCC are often similar to those caused by the underlying hepatic disease. It is very hard for physicians to distinguish signs and symptoms of HCC in contests charac‐ terized by an advanced liver disease. Advanced liver cancer can be responsible for accelerated liver functions deterioration caused by the intrahepatic tumor growth.

In this chapter, we review the clinical signs and symptoms induced by advanced carcinoma. We also discuss particular clinical scenarios caused by metastases and paraneoplastic syn‐ dromes, sometimes described case reports in literature.

### **2. Non-specific signs and symptoms**

Non-specific systemic signs and symptoms as asthenia, anorexia, weight loss, and nausea, are often present in patients with HCC (table 1). HCC should be suspected with the onset of these clinical features in patients at risk for this tumor.

© 2013 Barghini et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2.1. Clinical aspects of cirrhosis**

Clinical signs and symptoms of hepatic cirrhosis that is often present in patients with HCC, usually mask the presence of an underlying early hepatocellular carcinoma [1]. Symptoms and signs of cirrhosis are often the only expression of the disease. Because of this, patients affected by HCC usually present at an advanced stage of the disease with clinical signs as jaundice, ascites, peripheral oedemas, neurologic manifestations of hepatic encephalopathy, bleeding, or infections. Other signs of hepatic cirrhosis include gynecomastia, palmar erythema, spider angiomas, axillary or chest hair loss, hypogonadism (testicular atrophy, loss of libido).

in black South African patients, abdominal pain is frequent in 95% of cases while it is referred

Signs and Symptoms

199

http://dx.doi.org/10.5772/56162

Abdominal pain is more frequent in non cirrhotic patients, and in case of portal thrombosis [1]. Portal vein thrombosis has been found in (14-44%) of autopsies of patients with HCC [6,7]. Patients with both cirrhosis and hepatic carcinoma have the highest risk to develop portal vein thrombosis [6]. Portal vein thrombosis is reported to be diagnosed during investigation for

Other clinical manifestations of portal vein thrombosis and/or portal hypertension are hematemesis from rupture of esophageal varices, nausea, vomiting, anorexia, weight loss, diarrhea. Splenomegaly has been reported to be present in 75-100% of patients with portal vein thrombosis [9]. Bleeding from esophageal varices or from portal hypertensive gastropathy is the most common presenting symptom of portal vein thrombosis in cirrhotic patients [8,10]. In 43% of cases of Portal vein thrombosis in cirrhotic patients, diagnosis is done during a

If a cirrhotic patient present an acute pain, then bleeding from rupture of tumor should be suspected. HCC rupture causes a severe and sudden pain, and the patient can present the clinical features of an acute abdomen, with rebound and tenderness during physician palpa‐ tion and an abdominal involuntary defense contraction. A hypovolemic state and signs and symptoms of acute anemia can be present. Clinical features of chronic anemia can be present

HCC rupture has been reported to be a rare condition in Western countries, occurring only in 3% of Italian patients [11], while spontaneous hemoperitoneum is more frequent in Sub-Saharian Africa and in Southeast Asia, being present in 10% of patients at presentation [12]. Usually,HCC tumors bleedings are spontaneous, but in rare cases, they can be caused by external causes. The protrusion of HCC beyond the liver surface seems to be an important risk factor for HCC rupture [13]: hemorrhages occur more easily and can also be caused by slight external forces. So HCC rupture can be caused by abdominal traumas [14],vigorous muscular

HCC rupture is also a rare complication of therapeutic procedures on HCC. For example after transcatheter arterial chemoembolization (TACE), HCC rupture occurs in less than 1% of

The drainage of hematic peritoneal liquid in patient with acute abdominal pain can suggest

A particular manifestation of HCC can be a bleeding from esophageal varices. This presenta‐ tion is not frequent, occurring as first clinical sign only in 1%-8% of cases of HCC [17,18] (table1). Variceal bleeding is caused by higher pressure in portal district which in turn can be caused by tumor invasion of this venous system and portal hypertension [17]. This portal invasion can be detected radiologically in 44-57% [17,18] of cases of variceal bleeding as

by 46%, 51% and 38% of Japanese, Chinese and Italian patients, respectively (table 1).

acute abdominal pain in 18% of cases in cirrhotic patients [8].

exertion, or rarely after forceful physician's palpation [15].

the presence of a ruptured HCC, not being specific for this condition.

routine echo-Doppler examination [8].

in case of slow blood loss from HCC.

patients [16].

**3.1. Gastrointestinal bleeding**

A large HCC can worsen the underlying hepatic disease, therefore in case of clinical worsening of a cirrhotic patient, onset of a HCC should be suspected.

#### **2.2. Hepatomegaly**

Hepatomegaly can be an expression of the tumor mass,(table 1) [1,2]. In case of HCC, the palpable edge of the liver is more often irregular, hard, with nodular consistency.

Hepatomegaly is more often present in patients without advanced cirrhosis [2]. In case of large tumors, the mass can cause asymmetry of the abdomen [3]. The costal margin can be deformed and the profile of lower ribs can be asymmetric. The right hemi-diaphragm can be elevated. This alteration of diaphragm profile can be asymptomatic or can cause mild respiratory symptoms. With thorax percussion is possible to detect an area of dullness, while trough auscultation is possible not to hear the vescicular murmur in case of elevation of diaphragm. HCC can also cause a pleural effusion. All these clinical aspects are difficult to be differentiated from signs and symptoms of the underlying chronic liver disease: Right diaphragm elevation is possible in case of hepatopathy not associated to HCC and pleural effusion can be the expression of an ascending ascitic fluid or of the anasarca state caused by cirrhosis and hypoalbuminemia.

#### **2.3. Vascular bruit**

Trough auscultation of the abdomen, an arterial bruit can be heard in patients affected by HCC. This bruit is typically heard throughout the liver and it is described to have different charac‐ teristic from other vascular abdominal auscultatory findings. In fact, usually, arterial bruit caused by abdominal aortic aneurysm or by renal artery stenosis are soft and short. Arterial bruit caused by HCC is usually a hard bruit and it is more prolonged than those caused by other conditions. This clinical sign is thought to be caused by the presence of an arteriovenous fistula in the context of the tumor [4], suggesting the presence of a highly vascularized HCC [5].

### **3. Abdominal pain, portal vein thrombosis, rupture of HCC**

A frequent manifestation of onset of HCC is abdominal pain. The pain is usually mild, located in right hypochondrium and it can radiate to the right shoulder. Prior reports suggested that in black South African patients, abdominal pain is frequent in 95% of cases while it is referred by 46%, 51% and 38% of Japanese, Chinese and Italian patients, respectively (table 1).

Abdominal pain is more frequent in non cirrhotic patients, and in case of portal thrombosis [1]. Portal vein thrombosis has been found in (14-44%) of autopsies of patients with HCC [6,7]. Patients with both cirrhosis and hepatic carcinoma have the highest risk to develop portal vein thrombosis [6]. Portal vein thrombosis is reported to be diagnosed during investigation for acute abdominal pain in 18% of cases in cirrhotic patients [8].

Other clinical manifestations of portal vein thrombosis and/or portal hypertension are hematemesis from rupture of esophageal varices, nausea, vomiting, anorexia, weight loss, diarrhea. Splenomegaly has been reported to be present in 75-100% of patients with portal vein thrombosis [9]. Bleeding from esophageal varices or from portal hypertensive gastropathy is the most common presenting symptom of portal vein thrombosis in cirrhotic patients [8,10]. In 43% of cases of Portal vein thrombosis in cirrhotic patients, diagnosis is done during a routine echo-Doppler examination [8].

If a cirrhotic patient present an acute pain, then bleeding from rupture of tumor should be suspected. HCC rupture causes a severe and sudden pain, and the patient can present the clinical features of an acute abdomen, with rebound and tenderness during physician palpa‐ tion and an abdominal involuntary defense contraction. A hypovolemic state and signs and symptoms of acute anemia can be present. Clinical features of chronic anemia can be present in case of slow blood loss from HCC.

HCC rupture has been reported to be a rare condition in Western countries, occurring only in 3% of Italian patients [11], while spontaneous hemoperitoneum is more frequent in Sub-Saharian Africa and in Southeast Asia, being present in 10% of patients at presentation [12].

Usually,HCC tumors bleedings are spontaneous, but in rare cases, they can be caused by external causes. The protrusion of HCC beyond the liver surface seems to be an important risk factor for HCC rupture [13]: hemorrhages occur more easily and can also be caused by slight external forces. So HCC rupture can be caused by abdominal traumas [14],vigorous muscular exertion, or rarely after forceful physician's palpation [15].

HCC rupture is also a rare complication of therapeutic procedures on HCC. For example after transcatheter arterial chemoembolization (TACE), HCC rupture occurs in less than 1% of patients [16].

The drainage of hematic peritoneal liquid in patient with acute abdominal pain can suggest the presence of a ruptured HCC, not being specific for this condition.

#### **3.1. Gastrointestinal bleeding**

**2.1. Clinical aspects of cirrhosis**

198 Hepatocellular Carcinoma - Future Outlook

**2.2. Hepatomegaly**

hypoalbuminemia.

**2.3. Vascular bruit**

Clinical signs and symptoms of hepatic cirrhosis that is often present in patients with HCC, usually mask the presence of an underlying early hepatocellular carcinoma [1]. Symptoms and signs of cirrhosis are often the only expression of the disease. Because of this, patients affected by HCC usually present at an advanced stage of the disease with clinical signs as jaundice, ascites, peripheral oedemas, neurologic manifestations of hepatic encephalopathy, bleeding, or infections. Other signs of hepatic cirrhosis include gynecomastia, palmar erythema, spider angiomas, axillary or chest hair loss, hypogonadism (testicular atrophy, loss of libido).

A large HCC can worsen the underlying hepatic disease, therefore in case of clinical worsening

Hepatomegaly can be an expression of the tumor mass,(table 1) [1,2]. In case of HCC, the

Hepatomegaly is more often present in patients without advanced cirrhosis [2]. In case of large tumors, the mass can cause asymmetry of the abdomen [3]. The costal margin can be deformed and the profile of lower ribs can be asymmetric. The right hemi-diaphragm can be elevated. This alteration of diaphragm profile can be asymptomatic or can cause mild respiratory symptoms. With thorax percussion is possible to detect an area of dullness, while trough auscultation is possible not to hear the vescicular murmur in case of elevation of diaphragm. HCC can also cause a pleural effusion. All these clinical aspects are difficult to be differentiated from signs and symptoms of the underlying chronic liver disease: Right diaphragm elevation is possible in case of hepatopathy not associated to HCC and pleural effusion can be the expression of an ascending ascitic fluid or of the anasarca state caused by cirrhosis and

Trough auscultation of the abdomen, an arterial bruit can be heard in patients affected by HCC. This bruit is typically heard throughout the liver and it is described to have different charac‐ teristic from other vascular abdominal auscultatory findings. In fact, usually, arterial bruit caused by abdominal aortic aneurysm or by renal artery stenosis are soft and short. Arterial bruit caused by HCC is usually a hard bruit and it is more prolonged than those caused by other conditions. This clinical sign is thought to be caused by the presence of an arteriovenous fistula in the context of the tumor [4], suggesting the presence of a highly vascularized HCC [5].

A frequent manifestation of onset of HCC is abdominal pain. The pain is usually mild, located in right hypochondrium and it can radiate to the right shoulder. Prior reports suggested that

**3. Abdominal pain, portal vein thrombosis, rupture of HCC**

palpable edge of the liver is more often irregular, hard, with nodular consistency.

of a cirrhotic patient, onset of a HCC should be suspected.

A particular manifestation of HCC can be a bleeding from esophageal varices. This presenta‐ tion is not frequent, occurring as first clinical sign only in 1%-8% of cases of HCC [17,18] (table1). Variceal bleeding is caused by higher pressure in portal district which in turn can be caused by tumor invasion of this venous system and portal hypertension [17]. This portal invasion can be detected radiologically in 44-57% [17,18] of cases of variceal bleeding as presenting clinical manifestation of HCC. If variceal bleeding can be related with portal venous system invasion suggesting an advanced neoplastic disease, it does not seem to exist a relationship between this kind of clinical presentation and the size of the underlying tumor. Bleeding from esophageal varices is obviously a more frequent clinical presentation of HCC in patients with more advanced liver cirrhosis [18] and high degree of portal hypertension. However, HCC can present with variceal bleeding also in patients without a known history of hepatopathy [17].

**3.4. Caval invasion**

primary manifestation of HCC [30].

clinical aspect caused by HCC.

**Table 1.** [1,2,11,24]

**4. Age differences in presentation**

megaly [24].

If HCC invades the inferior vena cava, signs and symptoms of venous insufficiency can appear. In this case relevant pitting edema can appear, usually bilaterally, affecting both inferior limbs, from the inguinal region. The invasion of the venous district, can worsen ascites and hepato‐

Signs and Symptoms

201

http://dx.doi.org/10.5772/56162

Caval tumor thrombus can extend to the right atrium, causing dyspnea and heart failure [27]. When a patient presents signs and symptoms of right heart failure, such as jugular turgor, dyspnea, new onset of inferior limbs edema and worsening of hepatic insufficiency, heart tumoral invasion should always be suspected [28]. Anyway atrial invasion is reported to be also asymptomatic [29]. Pulmonary embolization by venous invasion is a rare, but reported

Patient's age can influence the clinical presentation of HCC. Signs and symptoms at presen‐ tation of HCC described in patients affected by hepatitis B are significantly different in patients younger and older than 40 years. Younger patients present more often with pain, hepatome‐ galy and ruptured HCC. Older patient present more often with ankle oedema and ascites. This is explained by the fact that in patients affected by viral hepatitis, advanced cirrhosis is more frequent in the older ones [2]. In younger patients it is more difficult that cirrhosis masks

**Clinical signs and symptoms in different geographic regions (%)**

Asymtomatic - - 29,9 38 Abdominal pain 95 46 51 38 Ascites 51 27 18 12 Palpable mass 92 23 5 - Hepatomegaly - - 54 90 Ankle Edema - 17 14 - Jaundice 28 17 9 14 Fever 35 17 2 12 Diarrhea - - 1 3 Hemoperitoneum - 7 3 3 Variceal Bleeding 2 8 - 4

**Black African Japan China Europe (Italy)**

Variceal bleeding can present with melena or hematemesis. Bleeding can be massive, leading to hypovolemic state and it is one of the known triggers of cirrhotic encephalopathy so that tremors, confusion till to coma, can be present in these patients too. Also infections can be caused by gastrointestinal bleeding in cirrhotic patients (22% of cases) [19], therefore, clinical signs and symptoms of an abdominal infections can be present.

Additionally, 50 % of causes of gastrointestinal hemorrhages are represented by hypertensive gastropathy, peptic ulcer and direct tumoral invasion of digestive tract [20,21].

#### **3.2. Jaundice**

Jaundice is a frequent sign of presentation of HCC. Some studies indicated that it is present at the diagnosis of HCC in 28% of African patients, but less frequent in Chinese, Japanese or European countries (table1).

Different pathologic conditions linked to HCC can explain the onset of jaundice. Jaundice can be expression of hepatic failure, due to extensive tumor infiltration of a cirrhotic liver or by worsening of the underlying hepatitis that can occur in presence of HCC. [22].

In other cases, jaundice result from obstruction of bile ducts by HCC. Clinical manifestation are those of typical cholestatic syndrome. In these cases jaundice is usually accompanied by itchiness, caused by elevation of serum level of bile acids, hypocolic stool and dark urine. All these symptoms can be presents also in the underlying liver disease, not being specific for biliary tract invasion.

The neoplastic obstruction can occur due to intraluminal biliary obstruction, extraluminal neoplastic compression or clot formation secondary to hemobilia caused by tumor invasion of biliary tree [23]. The presence of an intraluminal free-floating tumor fragment in the extrahe‐ patic biliary tree may show an intermittent jaundice that can be associated with colicky pain [24]. Also in case of hemobilia a colicky pain can be present [5].

#### **3.3. Fever**

Fever of Unknown Origin can be a way of presentation of HCC [25,26]. It can be intermittent, and usually is accompanied by leukocytosis. Imaging studies are often necessary to differen‐ tiate an HCC from a liver abscess. Fever occur more frequently in patients with massive HCC and in non cirrhotic individuals [1].

#### **3.4. Caval invasion**

presenting clinical manifestation of HCC. If variceal bleeding can be related with portal venous system invasion suggesting an advanced neoplastic disease, it does not seem to exist a relationship between this kind of clinical presentation and the size of the underlying tumor. Bleeding from esophageal varices is obviously a more frequent clinical presentation of HCC in patients with more advanced liver cirrhosis [18] and high degree of portal hypertension. However, HCC can present with variceal bleeding also in patients without a known history

Variceal bleeding can present with melena or hematemesis. Bleeding can be massive, leading to hypovolemic state and it is one of the known triggers of cirrhotic encephalopathy so that tremors, confusion till to coma, can be present in these patients too. Also infections can be caused by gastrointestinal bleeding in cirrhotic patients (22% of cases) [19], therefore, clinical

Additionally, 50 % of causes of gastrointestinal hemorrhages are represented by hypertensive

Jaundice is a frequent sign of presentation of HCC. Some studies indicated that it is present at the diagnosis of HCC in 28% of African patients, but less frequent in Chinese, Japanese or

Different pathologic conditions linked to HCC can explain the onset of jaundice. Jaundice can be expression of hepatic failure, due to extensive tumor infiltration of a cirrhotic liver or by

In other cases, jaundice result from obstruction of bile ducts by HCC. Clinical manifestation are those of typical cholestatic syndrome. In these cases jaundice is usually accompanied by itchiness, caused by elevation of serum level of bile acids, hypocolic stool and dark urine. All these symptoms can be presents also in the underlying liver disease, not being specific for

The neoplastic obstruction can occur due to intraluminal biliary obstruction, extraluminal neoplastic compression or clot formation secondary to hemobilia caused by tumor invasion of biliary tree [23]. The presence of an intraluminal free-floating tumor fragment in the extrahe‐ patic biliary tree may show an intermittent jaundice that can be associated with colicky pain

Fever of Unknown Origin can be a way of presentation of HCC [25,26]. It can be intermittent, and usually is accompanied by leukocytosis. Imaging studies are often necessary to differen‐ tiate an HCC from a liver abscess. Fever occur more frequently in patients with massive HCC

gastropathy, peptic ulcer and direct tumoral invasion of digestive tract [20,21].

worsening of the underlying hepatitis that can occur in presence of HCC. [22].

[24]. Also in case of hemobilia a colicky pain can be present [5].

signs and symptoms of an abdominal infections can be present.

of hepatopathy [17].

200 Hepatocellular Carcinoma - Future Outlook

**3.2. Jaundice**

European countries (table1).

biliary tract invasion.

and in non cirrhotic individuals [1].

**3.3. Fever**

If HCC invades the inferior vena cava, signs and symptoms of venous insufficiency can appear. In this case relevant pitting edema can appear, usually bilaterally, affecting both inferior limbs, from the inguinal region. The invasion of the venous district, can worsen ascites and hepato‐ megaly [24].

Caval tumor thrombus can extend to the right atrium, causing dyspnea and heart failure [27]. When a patient presents signs and symptoms of right heart failure, such as jugular turgor, dyspnea, new onset of inferior limbs edema and worsening of hepatic insufficiency, heart tumoral invasion should always be suspected [28]. Anyway atrial invasion is reported to be also asymptomatic [29]. Pulmonary embolization by venous invasion is a rare, but reported primary manifestation of HCC [30].

### **4. Age differences in presentation**

Patient's age can influence the clinical presentation of HCC. Signs and symptoms at presen‐ tation of HCC described in patients affected by hepatitis B are significantly different in patients younger and older than 40 years. Younger patients present more often with pain, hepatome‐ galy and ruptured HCC. Older patient present more often with ankle oedema and ascites. This is explained by the fact that in patients affected by viral hepatitis, advanced cirrhosis is more frequent in the older ones [2]. In younger patients it is more difficult that cirrhosis masks clinical aspect caused by HCC.


**Table 1.** [1,2,11,24]

### **5. Extrahepatic metastases**

Metastases from HCC, spread through lymphatic or hematic system, are more frequently placed in abdominal and thoracic lymph nodes, lung, bones, adrenal glands. Less frequent sites of metastases are brain, spleen and breast [31]. Rarely metastases can also be detected in digestive tube, pancreas, seminal vesicle and bladder [32].

When HCC is diagnosed, extrahepatic metastases, are present in more or less 40% of cases, [32,33] and several signs and symptoms can be caused by this condition. Sometimes signs and symptoms caused by metastases are the only clinical manifestation of HCC [34]. Regional lymph nodes are affected in up to 60% of metastatic HCC, while distant lymphatic stations are involved only in 12% of these cases [32]. The frequencies of metastases in different sites are reported in (Table 2).

Pain and pathologic fractures can be caused by osteolytic metastases. Severe pain is present in 90 % of patients with bone metastases [35]. Bone metastases are present in up 66% of patients in some studies, mostly in the transverse skeleton as in thoracic spine, lumbosacral spine, sacrum. Frequently they can also be present in ribs, skull, head of femur and peripheral bones [24,32]. Rarely HCC can have as only presentation pain or other symptoms caused by bone metastases, and this can also occur in non-common bone sites [36,37].

hypoglycemia and hypercalcemia are some of the most common paraneoplastic manifestations of HCC. Only 7% of patients have 2 of these syndromes, and rarely can be present all of them

Seminal Vescicle 1 Bladder 1

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**Frequency of metastases in different sites (%) Frequent Sites Not Frequent Sites**

Lungs 55 Brain 2 Lymph Nodes 53 Rectum 1 Regional 41 Spleen 1 Distant 12 Diaphragm 1 Musculoskeletal 28 Duodenum 1 Adrenal 11 Esophagus 1 Peritoneum and/or omenutm 11 Pancreas 1

Below, there are described the most common paraneoplastic syndromes and there are also reported other manifestations described in some case reports. See (Table 3) for some of the

Hypoglycemia is not an infrequent paraneoplastic syndrome caused by HCC, occurring in about 4,6-27% of the patients with advanced HCC [43–45]. Hypoglycemia is caused by the increased demand for glucose by the tumor together with a reduction of gluconeogenesis and glycogenolysis, due to a decreased residual liver tissue coupled with cachectic state and malnutrition that are often present in these patients [46]. Episodes of hypoglycemia caused by

Another pathologic mechanism involved in causing hypoglycemia is overproduction by the tumor of Insulin-Like Growth Factor II [47]. Hypoglycemia caused by over production of IGF II occurs not only in late stage HCC, but also in the early phases of the neoplastic disease [44].

Hypoglycemia has been also described as the first clinical manifestation of HCC in some

Hypercalcemia is described to occur in 7-12% of patients with HCC [43,45,49]. A PTH-like humoral factor is probably responsible of this paraneoplastic syndrome [50]. Hypercalcemia

can also be caused by the presence of osteolytic bone metastases [51].

an advanced HCC typically occur in the last weeks before the death of the patients.

(<1% of cases) [42].

**Table 2.** [32]

**6.1. Hypoglycemia**

reports [44,48].

**6.2. Hypercalcemia**

reported paraneoplastc syndromes.

HCC metastases can cause also symptoms linked to nervous system. In fact in case of vertebral fractures, spinal cord compression can occur, causing neurological symptoms, leading to paraplegia in some cases. Clinical features of spinal compression can occur as complication of advanced known HCC, or rarely they can be the clinical presentation of this tumor [38].

Excluding lymph nodes, lung is the most common site of metastases (54% of metastatic HCC) [39].Lung metastases sometimes are causes of dyspnea, cough, hemoptysis, chest pain [40]. Fatal respiratory failure is described in more or less 20% of HCC lung metastatic tumors [39].

Brain metastases are not frequent, but often they cause important neurological symptoms, up to causing paralysis in most of these cases [39].

Peritoneal metastases can cause ascites and abdominal pain and they are present in more or less 10% of metastatic HCC [32]. Rarely, metastases were reported in appendix, and signs and symptoms typical of acute appendicitis, as pain in the right lower quadrant associated with tenderness at the physical examination, can be a clinical presentation of HCC [41].

### **6. Paraneoplastic syndromes**

Paraneoplastic syndromes occur in 19-44% of patients affected by HCC [42,43]. The presence of paraneoplastic syndromes is described to be related with younger patients, with larger size tumor (>10 cm) and with presence of portal vein thrombosis [43].

Among patients that have paraneoplastic syndromes during the clinical course of HCC, most of them have a single paraneoplastic manifestation. Hypercholesterolemia, erythrocytosis,


#### **Table 2.** [32]

**5. Extrahepatic metastases**

202 Hepatocellular Carcinoma - Future Outlook

reported in (Table 2).

digestive tube, pancreas, seminal vesicle and bladder [32].

metastases, and this can also occur in non-common bone sites [36,37].

to causing paralysis in most of these cases [39].

**6. Paraneoplastic syndromes**

Metastases from HCC, spread through lymphatic or hematic system, are more frequently placed in abdominal and thoracic lymph nodes, lung, bones, adrenal glands. Less frequent sites of metastases are brain, spleen and breast [31]. Rarely metastases can also be detected in

When HCC is diagnosed, extrahepatic metastases, are present in more or less 40% of cases, [32,33] and several signs and symptoms can be caused by this condition. Sometimes signs and symptoms caused by metastases are the only clinical manifestation of HCC [34]. Regional lymph nodes are affected in up to 60% of metastatic HCC, while distant lymphatic stations are involved only in 12% of these cases [32]. The frequencies of metastases in different sites are

Pain and pathologic fractures can be caused by osteolytic metastases. Severe pain is present in 90 % of patients with bone metastases [35]. Bone metastases are present in up 66% of patients in some studies, mostly in the transverse skeleton as in thoracic spine, lumbosacral spine, sacrum. Frequently they can also be present in ribs, skull, head of femur and peripheral bones [24,32]. Rarely HCC can have as only presentation pain or other symptoms caused by bone

HCC metastases can cause also symptoms linked to nervous system. In fact in case of vertebral fractures, spinal cord compression can occur, causing neurological symptoms, leading to paraplegia in some cases. Clinical features of spinal compression can occur as complication of advanced known HCC, or rarely they can be the clinical presentation of this tumor [38].

Excluding lymph nodes, lung is the most common site of metastases (54% of metastatic HCC) [39].Lung metastases sometimes are causes of dyspnea, cough, hemoptysis, chest pain [40]. Fatal respiratory failure is described in more or less 20% of HCC lung metastatic tumors [39]. Brain metastases are not frequent, but often they cause important neurological symptoms, up

Peritoneal metastases can cause ascites and abdominal pain and they are present in more or less 10% of metastatic HCC [32]. Rarely, metastases were reported in appendix, and signs and symptoms typical of acute appendicitis, as pain in the right lower quadrant associated with

Paraneoplastic syndromes occur in 19-44% of patients affected by HCC [42,43]. The presence of paraneoplastic syndromes is described to be related with younger patients, with larger size

Among patients that have paraneoplastic syndromes during the clinical course of HCC, most of them have a single paraneoplastic manifestation. Hypercholesterolemia, erythrocytosis,

tenderness at the physical examination, can be a clinical presentation of HCC [41].

tumor (>10 cm) and with presence of portal vein thrombosis [43].

hypoglycemia and hypercalcemia are some of the most common paraneoplastic manifestations of HCC. Only 7% of patients have 2 of these syndromes, and rarely can be present all of them (<1% of cases) [42].

Below, there are described the most common paraneoplastic syndromes and there are also reported other manifestations described in some case reports. See (Table 3) for some of the reported paraneoplastc syndromes.

#### **6.1. Hypoglycemia**

Hypoglycemia is not an infrequent paraneoplastic syndrome caused by HCC, occurring in about 4,6-27% of the patients with advanced HCC [43–45]. Hypoglycemia is caused by the increased demand for glucose by the tumor together with a reduction of gluconeogenesis and glycogenolysis, due to a decreased residual liver tissue coupled with cachectic state and malnutrition that are often present in these patients [46]. Episodes of hypoglycemia caused by an advanced HCC typically occur in the last weeks before the death of the patients.

Another pathologic mechanism involved in causing hypoglycemia is overproduction by the tumor of Insulin-Like Growth Factor II [47]. Hypoglycemia caused by over production of IGF II occurs not only in late stage HCC, but also in the early phases of the neoplastic disease [44].

Hypoglycemia has been also described as the first clinical manifestation of HCC in some reports [44,48].

#### **6.2. Hypercalcemia**

Hypercalcemia is described to occur in 7-12% of patients with HCC [43,45,49]. A PTH-like humoral factor is probably responsible of this paraneoplastic syndrome [50]. Hypercalcemia can also be caused by the presence of osteolytic bone metastases [51].

Hypercaclemic coma can occur in patients with HCC and it can be confused with hepatic encephalopathy [52].

**6.8. Feminization**

literature, [82–84].

for HCC [87].

proximal extremities [85,86].

**8. Neurological manifestations**

presenting manifestation of HCC [93].

resis and dysarthria, also in young patients [91,92].

feminization is still not defined [71–74].

HCC, sometimes causing severe rhabdomyolysis [78–81].

[88]. This sign has been reported also in association to HCC [89]. Also paraneoplastic pemphigus has been related to HCC [90].

**7. Cutaneous manifestaion**

Feminization is described to be present in patient with HCC, also in those not affected by liver cirrhosis, showing clinical signs and symptoms as gynecomastia, loss of body hair, loss of libido. Onset of spider naevi can be a sign of an underlying HCC and this finding has been reported more frequently in men than in women [70]. Studies about circulating sex hormones in patients affected by HCC have shown unclear results and a direct role of HCC in causing

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Various cutaneous manifestations have been reported as paraneoplastic syndromes caused by HCC. In rare cases, dermatomyositis has been described in patients affected by HCC, either associated or not to viral hepatitis. [75–77]. Also polymyositis can be a paraneoplastic mani‐ festation of HCC. Polymyositis has been described also as a rare presenting manifestation of

A relationship between HCC and acquired porphyria cutanea tarda has been reportedin

Pityriasis rotunda is a rare cutaneous disease characterized by round/oval, hypo/hyperpig‐ mented rash with scaling. The number of skin lesion is variable and the size is described to be between 1,5 to 25 cm. The localization of the lesions is usually over the trunk, lower back and

In South African Black patients with diagnosis of HCC, its prevalence is of 16%, much higher than in patients affected by other diseases associated with pityriasis rotunda. In selected groups of people, the onset of this cutaneous disorder could represent a diagnostic instrument

The sign of Leser-Trelat defined as the abrupt appearance and rapid increase in size and number of multiple seborrheic keratoses as a result of cancer development has been reported

Neurologic paraneoplasic syndromes are rarely present in patients affected by HCC, such as multifocal necrotizing leukoencephalopathy leading to coma or non-inflammatory cerebral vasculopathy with widespread cortical and subcortical infarcts causing progressive hemipa‐

Peripheral polyneuropathy with cranial nerve involvement has been described as a rare

#### **6.3. Hypercholesterolemia**

Paraneoplastic hypercholesterolemia is present in 11-20% of patients with HCC [43,45,53]. Hypercholesterolemia in patients with HCC has been related with a reduced expression of LDL receptor, as in familiar hypercholesterolemia. Clinical manifestation of hypercholestero‐ lemia in patients with HCC are not reported, but increased cardiovascular risk can be present and manifestations typical of familiar hypercholesterolemia, as xanthelasmas could be possible.

#### **6.4. Erythrocytosis**

Erythrocytosis is present in 2-16% of patients with HCC [45,54–58]. Higher levels of erythro‐ poietin, produced by the tumor, have been reported [58]. Local hypoxia has been suggested to be the cause of overproduction of erythropoietin by large HCC tumors [58,59].

Erythrocytosis accompanied by high levels of erythropoietin has been described as a rare primary presentation of HCC [60].

#### **6.5. Thrombocytosis**

Thrombocytosis has been reported to be present in 3% of cases of HCC. This paraneoplastic syndrome seems to occur in younger patients (<60 yr), being related with the presence of a larger tumor and main portal vein tumor thrombosis representing an unfavorable prognostic factor [61]. Thrombocytosis has been described to be related with thrombopoietin production by the HCC [62]. A case of HCC associated with both thrombocytosis and acquired Von Willebrand disease has been reported [63].

#### **6.6. Arterial hypertension**

Arterial hypertension has been described as a paraneoplastic manifestation of HCC. Some cases of severe arterial blood pressure associated with high plasma level of angiotensin-I, accompanied with hypokalemia have been reported [64]. Elevated concentrations of angio‐ tensinogen have been found, whether or not associated with higher plasma levels of renin. Overproduction of angiotensinogen and renin could both play a role in causing paraneoplastic hypertension in patients affected by HCC [65].

#### **6.7. Diarrhea**

Watery diarrhea is another manifestation that can occur in patients with HCC. Overproduction of intestinal peptides as gastrin and vasoactive intestinal peptide (VIP) is one possible explanation of the onset of diarrhea in these patients [66]. Diarrhea was also described to be a possible clinical presentation of HCC [67–69].

#### **6.8. Feminization**

Hypercaclemic coma can occur in patients with HCC and it can be confused with hepatic

Paraneoplastic hypercholesterolemia is present in 11-20% of patients with HCC [43,45,53]. Hypercholesterolemia in patients with HCC has been related with a reduced expression of LDL receptor, as in familiar hypercholesterolemia. Clinical manifestation of hypercholestero‐ lemia in patients with HCC are not reported, but increased cardiovascular risk can be present and manifestations typical of familiar hypercholesterolemia, as xanthelasmas could be

Erythrocytosis is present in 2-16% of patients with HCC [45,54–58]. Higher levels of erythro‐ poietin, produced by the tumor, have been reported [58]. Local hypoxia has been suggested

Erythrocytosis accompanied by high levels of erythropoietin has been described as a rare

Thrombocytosis has been reported to be present in 3% of cases of HCC. This paraneoplastic syndrome seems to occur in younger patients (<60 yr), being related with the presence of a larger tumor and main portal vein tumor thrombosis representing an unfavorable prognostic factor [61]. Thrombocytosis has been described to be related with thrombopoietin production by the HCC [62]. A case of HCC associated with both thrombocytosis and acquired Von

Arterial hypertension has been described as a paraneoplastic manifestation of HCC. Some cases of severe arterial blood pressure associated with high plasma level of angiotensin-I, accompanied with hypokalemia have been reported [64]. Elevated concentrations of angio‐ tensinogen have been found, whether or not associated with higher plasma levels of renin. Overproduction of angiotensinogen and renin could both play a role in causing paraneoplastic

Watery diarrhea is another manifestation that can occur in patients with HCC. Overproduction of intestinal peptides as gastrin and vasoactive intestinal peptide (VIP) is one possible explanation of the onset of diarrhea in these patients [66]. Diarrhea was also described to be a

to be the cause of overproduction of erythropoietin by large HCC tumors [58,59].

encephalopathy [52].

possible.

**6.4. Erythrocytosis**

**6.5. Thrombocytosis**

**6.6. Arterial hypertension**

**6.7. Diarrhea**

primary presentation of HCC [60].

Willebrand disease has been reported [63].

hypertension in patients affected by HCC [65].

possible clinical presentation of HCC [67–69].

**6.3. Hypercholesterolemia**

204 Hepatocellular Carcinoma - Future Outlook

Feminization is described to be present in patient with HCC, also in those not affected by liver cirrhosis, showing clinical signs and symptoms as gynecomastia, loss of body hair, loss of libido. Onset of spider naevi can be a sign of an underlying HCC and this finding has been reported more frequently in men than in women [70]. Studies about circulating sex hormones in patients affected by HCC have shown unclear results and a direct role of HCC in causing feminization is still not defined [71–74].

### **7. Cutaneous manifestaion**

Various cutaneous manifestations have been reported as paraneoplastic syndromes caused by HCC. In rare cases, dermatomyositis has been described in patients affected by HCC, either associated or not to viral hepatitis. [75–77]. Also polymyositis can be a paraneoplastic mani‐ festation of HCC. Polymyositis has been described also as a rare presenting manifestation of HCC, sometimes causing severe rhabdomyolysis [78–81].

A relationship between HCC and acquired porphyria cutanea tarda has been reportedin literature, [82–84].

Pityriasis rotunda is a rare cutaneous disease characterized by round/oval, hypo/hyperpig‐ mented rash with scaling. The number of skin lesion is variable and the size is described to be between 1,5 to 25 cm. The localization of the lesions is usually over the trunk, lower back and proximal extremities [85,86].

In South African Black patients with diagnosis of HCC, its prevalence is of 16%, much higher than in patients affected by other diseases associated with pityriasis rotunda. In selected groups of people, the onset of this cutaneous disorder could represent a diagnostic instrument for HCC [87].

The sign of Leser-Trelat defined as the abrupt appearance and rapid increase in size and number of multiple seborrheic keratoses as a result of cancer development has been reported [88]. This sign has been reported also in association to HCC [89].

Also paraneoplastic pemphigus has been related to HCC [90].

### **8. Neurological manifestations**

Neurologic paraneoplasic syndromes are rarely present in patients affected by HCC, such as multifocal necrotizing leukoencephalopathy leading to coma or non-inflammatory cerebral vasculopathy with widespread cortical and subcortical infarcts causing progressive hemipa‐ resis and dysarthria, also in young patients [91,92].

Peripheral polyneuropathy with cranial nerve involvement has been described as a rare presenting manifestation of HCC [93].

### **9. Other paraneoplastic syndromes**

Other paraneoplastic manifestations rarely described in patients with HCC are dysfibrino‐ gennemia [94,95], cryofibrinogenemia [96], carcinoid syndrome [24], myasthenia gravis [97] and membranous glomerulonephritis [98].

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**Table 3.** Add caption

#### **10. Conclusion**

Clinical signs and symptoms of HCC are various. In absence of screening programs clinical signs can be helpful for diagnosis. Clinical signs are different depending on tumor size, vascular invasion, presence of cirrhosis and presence of metastases. Several paraneoplastic syndromes are described but our knowledge about some manifestation is limited to few published case reports.

#### **Author details**

Valerio Barghini, Debora Donnini, Alessandro Uzzau\* and Giorgio Soardo\*

\*Address all correspondence to: soardo.giorgio@aoud.sanita.fvg.it

Department of Clinical and Experimental Sciences, University of Udine, Italy

### **References**

**9. Other paraneoplastic syndromes**

206 Hepatocellular Carcinoma - Future Outlook

and membranous glomerulonephritis [98].

Thrombocytosis Pityriasis Rotunda

Arterial Hypertension Dermatomyositis/

Valerio Barghini, Debora Donnini, Alessandro Uzzau\*

\*Address all correspondence to: soardo.giorgio@aoud.sanita.fvg.it

Department of Clinical and Experimental Sciences, University of Udine, Italy

**Table 3.** Add caption

**10. Conclusion**

published case reports.

**Author details**

Other paraneoplastic manifestations rarely described in patients with HCC are dysfibrino‐ gennemia [94,95], cryofibrinogenemia [96], carcinoid syndrome [24], myasthenia gravis [97]

**Paraneoplastic Syndromes**

**Usual Manifestations**

Hypoglycemia Hypercalcemia Hypercholesterolemia Erythrocytosis

**Reported Manifestations**

Dysfibrinogenemia Cryofibrinogenemia Carcinoid syndrome Myasthenia grave

Clinical signs and symptoms of HCC are various. In absence of screening programs clinical signs can be helpful for diagnosis. Clinical signs are different depending on tumor size, vascular invasion, presence of cirrhosis and presence of metastases. Several paraneoplastic syndromes are described but our knowledge about some manifestation is limited to few

Sign of Leser-Trelat Diarrhea Neurological manifestations

Polymyositis Porphyria cutanea tarda Pemphigus

and Giorgio Soardo\*

Membranous glomerulonephritis


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**Chapter 11**

**Sorafenib in the Continuum of Care for Hepatocellular**

Globally, liver cancer represents a major health care burden, accounting for almost 700,000 deaths annually. [1] Hepatocellular carcinoma (HCC) comprises 70% to 85% of primary liver cancers in most regions. [2] Although the incidence of HCC has historically been lower in the US than in many other countries, age-adjusted rates tripled between 1975 and 2005. [3] In fact, liver cancer is the fastest-growing cause of cancer-related death in American men. [4] Most patients with HCC are diagnosed at advanced stages and are ineligible for potentially curative treatments such as surgical resection and liver transplantation. [5] Prior to the introduction of

In defining optimal treatment for patients with HCC, several questions remain unanswered. Clinical data are needed to evaluate sorafenib safety in patients with advanced liver disease (i.e., those with Child-Pugh [CP] B disease) and in those with HCC-associated portal hyper‐ tension. In addition, how best to utilize sorafenib in combination with sequential locoregional therapies (LRT) or post-surgery remains unclear. These challenges are further compounded by the ongoing uncertainties in determining the value of the modified Response Evaluation Criteria In Solid Tumors (mRECIST) and understanding the optimal timing for response assessment in patients treated with sorafenib and/or LRT. Proactive management of adverse events (AEs) associated with sorafenib also remains an area of active investigation. Finally, although sorafenib has demonstrated a clear benefit to patients with advanced HCC, the majority of patients will ultimately experience disease progression. Efforts are underway to evaluate the best approaches to treating these patients in a manner that minimizes the risk of mortality from deterioration of the underlying liver disease. In this review, we describe the current understanding of sorafenib's efficacy and safety, and ongoing approaches to defining

> © 2013 Kaseb and Kulik; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

sorafenib in 2007, systemic treatments were unavailable for patients with HCC.

**Carcinoma: Challenges in Defining Optimal Practice**

Ahmed O. Kaseb and Laura M. Kulik

http://dx.doi.org/10.5772/56952

**1. Introduction**

Additional information is available at the end of the chapter

even better treatment options for patients with HCC.
