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Carcinogenesis Advance Access originally published online on May 29, 2008
Carcinogenesis 2008 29(7):1299-1305; doi:10.1093/carcin/bgn113
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Recent advances in the natural history of hepatocellular carcinoma

F. Trevisani*, M.C. Cantarini, J.R. Wands1 and M. Bernardi

Dipartimento di Medicina Clinica, Università di Bologna, via Albertoni 15, 40138 Bologna, Italy
1 Liver Research Center, Rhode Island Hospital, The Warren Alpert Medical School of Brown University, 55 Claverick Street, 4th floor, Providence, RI 02903, USA

* To whom correspondence should be addressed. Dipartimento di Medicina Clinica, Unità di Semeiotica Medica, via Albertoni 15, 40138 Bologna, Italy., Tel: +39 051 636 2923;, Fax: +39 051 636 2930;, Email: franco.trevisani{at}unibo.it


   Abstract
Top
 Abstract
Introduction
 References
 
Ongoing advances in liver disease management and basic research in recent years have changed our knowledge of the natural history of hepatocellular carcinoma (HCC). Indeed, the natural history of this tumor is fairly long and covers a preclinical and a clinical phase. Some of the biological steps involved in cell transformation and different carcinogenic pathways have been identified, disclosing potential novel markers for HCC. Following the progress in surveillance and early diagnosis, much more is now known about precancerous lesions and the process leading to overt HCC, including growth patterns, dedifferentiation and neoangiogenenesis. In particular, research has focused on clinical and biological factors predicting tumor aggressiveness and patients’ prognosis. Lastly, clinical studies have described tumor presentation, evolution and causes of patients’ death and how the new knowledge has influenced clinical management and patients’ survival in recent years. By addressing 10 key questions, this review will summarize well-established and novel features of the natural history of HCC.

Abbreviations: AFP, alfa-fetoprotein; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HGDN, high-grade dysplastic nodule; IGF, insulin-like growth factor; MRN, macroregenerative nodule


   Introduction
Top
 Abstract
 Introduction
References
 
Hepatocellular carcinoma (HCC) is one of the commonest malignancies with >500 000 new tumors diagnosed annually (1). HCC usually arises in the setting of chronic liver diseases, mostly related to viral hepatitis B and C. Its incidence varies widely among the different geographic areas (from 2 to almost 50 per 100 000 males/year), reaching peak values in Southeast Asia and sub-Saharan Africa, where hepatitis B virus (HBV) infection is endemic. Nonetheless, the HCC incidence has been increasing in Western countries in recent years due to the spread of hepatitis C virus (HCV) infection in the 1960s and 1970s (2). The ‘natural history’ of a disease refers to its course from the biological changes marking disease onset to the patient's death. Ongoing advances in liver disease research have added new elements to our knowledge of the natural history of HCC. By addressing 10 key questions, this review will summarize well-established and novel features of the topic.

Question 1: what is the natural history of HCC, and has it changed in the last few decades?
Previously, HCC was invariably diagnosed at a late stage with the development of clinical symptoms and was subsequently characterized by a rapidly fatal course. Following major advances in diagnostic techniques and the establishment of surveillance programs, early tumors have been increasingly detected, leading to an important advance in our understanding of the natural history of HCC. Moreover, examination of liver removed from patients undergoing transplantation has provided the opportunity to identify and analyze precancerous lesions and minute tumors. Lastly, progress in molecular biology has defined some of the biological steps involved in cell transformation and different carcinogenic pathways leading to overt HCC. As a result, we now know that the natural history of HCC is fairly long and can be split into three distinct phases: (i) ‘molecular’; (ii) ‘preclinical’ and (iii) ‘clinical or symptomatic’ (Figure 1).


Figure 1
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  		Fig. 1. The natural history of HCC can be divided into three distinct phases: (i) molecular, (ii) preclinical and (iii) clinical or symptomatic. The preclinical phase covers an initial period, in which the tumor is too small to be detected by imaging techniques, and a second period (preclinical diagnostic phase), during which the tumor is detectable but still asymptomatic. Finally, the clinical or symptomatic phase starts with the occurrence of symptoms caused by the tumor burdens. In patients with chronic liver disease, HCC usually becomes symptomatic when it reaches 4.5–8 cm.

 
The molecular phase includes the sequential genomic alterations leading to cell transformation. It has been postulated that the transformed cell can be either a hepatocyte/biliary epithelial or a liver stem cell (3). The genetic alterations involving differentiated cells (hepatocytes and biliocytes) are thought to confer a growth advantage by promoting proliferation and inhibiting apoptosis, whereas those involving stem cells interfere with the differentiation process. In human carcinogenesis, the time to acquire these genetic changes is unknown.

The preclinical phase covers an initial period, in which the tumor is still too small to be detected by imaging techniques, and a second period (‘preclinical diagnostic’ phase), during which the tumor is detectable but still asymptomatic.

Finally, the clinical or symptomatic phase starts with the occurrence of symptoms caused by the tumor burden: in patients with chronic liver disease, HCC usually becomes symptomatic when it reaches 4.5–8 cm (4,5).

Retrospective cohort studies focused on HCV-infected patients suggest that the development of HCC requires ~10 years from the diagnosis of cirrhosis and ~30 years from exposure to HCV (6). The temporal sequence has not been defined for the other main risk factors, but a similar time course may be applicable to alcoholic liver disease. Conversely, the time course of HBV-related carcinogenesis is less predictable since HCC may precede the occurrence of cirrhosis, particularly with chronic HBV infection in endemic areas (7).

Question 2: what is the sequence of mutations leading to hepatic transformation?
During the ‘preneoplastic’ phase (chronic hepatitis and cirrhosis), genetic alterations are almost entirely ‘quantitative’, occurring by epigenetic mechanisms without changes in the structure of genes. In this phase, hepatocytes undergo an intense mitogenic stimulation due to exposure to elevated levels of growth factors [such as insulin-like growth factor (IGF)-2 and transforming growth factor-{alpha}] as well as inflammatory cytokines, which may lead to activation of the major signaling pathways involved in cell proliferation. The enhanced expression of growth factors and cytokines is driven by inflammation, the action of viral proteins and regenerative response to cell loss. The mechanisms whereby these factors affect gene expression include cis- and trans-activation and altered chromatin methylation and acetylation, with consequent activation or inactivation of gene promoters (8). Moreover, viral proteins, such as the protein X (HBX) produced by HBV, can directly stimulate the major cytosolic kinase signaling cascades (9–11).

Hepatocyte proliferation rate, telomere shortening and telomerase reexpression progressively increase from the preneoplastic phase to dysplasia and HCC (8).

‘Structural’ alterations of genes slowly develop during the preneoplastic phase and significantly increase in dysplastic and neoplastic hepatocytes. They can result from multiple mechanisms: (i) HBV is directly mutagenic following integration of the viral genome or fragments into the cellular DNA; (ii) molecular products of both HBV (HBX) and HCV (core, NS5A and NS3) may impair the function of the tumor suppressor p53 and retinoblastoma genes, and alter the efficiency of enzymes involved in gene repair and stability (8,12); (iii) erosion of telomere length in highly replicative cells results in chromosome disruption and mitotic alteration; (iv) oxidative DNA damage may occur in the setting of chronic inflammation (8,11). Finally, HBV genotoxicity is enhanced by exposure to aflatoxin B, a contaminating mycotoxin found in food in certain regions of the world (13).

Genomic alterations in HCC are very heterogeneous, suggesting that the neoplastic phenotype can result from different ‘genomic routes’. Indeed, multiple genes and loci alterations have been documented in HCC cells but any single aberration has a low prevalence in tumors (8). Nonetheless, recurring genomic losses or gains on some chromosome arms have been documented (losses: 1p, 4q, 5q, 6q, 8p, 9p, 13q, 16p, 16q and 17p; gains: 1p, 6p, 7q, 8q and 17q); some of the loci affected by the recurrent chromosome losses contain well-known tumor suppressor genes, such as p53 on 17p, retinoblastoma on 13q, axin1 on 16p, Cdkn2A (p16INK4) on 9p and IGF-2-receptor on 6q, which often undergo allelic deletion (8,10,13,14). In addition, gains may involve certain oncogenes, such as c-myc which is contained in a chromosomal region frequently amplified in HCC (8,10,13).

As a result, many genetic and epigenetic aberrations and the corresponding alterations in molecular pathways have been found during hepatocarcinogenesis: (i) inactivation of the tumor suppressor gene p53 through gene mutation and posttranscriptional interaction with viral proteins (10,11,13,14); (ii) activation of the Wnt/Frizzled/β-catenin pathway through mutations in β-catenin or in other components of its destruction complex (glycogen synthase kinase-β/adenomatous polyposis coli protein/axin) as well as through upregulation of upstream elements such as Frizzled receptors (9,10,13,14); (iii) alteration of the tumor suppressor retinoblastoma and p16INK4 genes through mutations or promoter methylation (10,13,14); (iv) alteration of the IGFs/IRS/MAPK signaling pathway through IGFs overexpression, IRS overexpression (9) and, probably, IGF-2 receptor mutations (10,13) and (v) alteration of the transforming growth factor-β pathway (10,13). More recently, activation of phosphatidyinositol 3-kinase/AKT pathway and activation of the janus kinase/signal transducer and activator of transcription pathway through aberrant methylation of suppressor of cytokine signaling genes have been also identified (10,13). Moreover, upregulation of genes involved in angiogenesis, such as vascular endothelial growth factor, and genes in cell dissemination and metastases, such as matrix metalloproteinases, have been shown to play a role in hepatocarcinogenesis (14).

Finally, the recent microarray techniques have identified the ‘genetic profiles’ that reflect progression from preneoplastic lesions to early and advanced HCC (15): many genes were differentially expressed at each stage of the disease, some of which may be potential novel markers for HCC. In particular, the development of HCC is associated with changes in the expression of gene regulating the immune response in the early stages, whereas an upregulation of genes controlling DNA replication and cell cycle can be found in the late stages of disease.

Question 3: what are the histological ‘precancerous’ lesions in hepatocarcinogenesis?
Cirrhosis is the substrate of HCC in 80–90% of cases. In this setting, HCC can develop inside macroregenerative nodules (MRNs). This type of carcinogenesis is called ‘nodule in nodule’ (Figure 2) (16). The MRNs are detectable in 25% of cirrhotic livers as nodules macroscopically distinguishable from the surrounding liver in terms of color, size (diameter >0.5 cm, usually 1–2 cm) and tissue texture (17). The histological structure resembles that of cirrhotic nodules, although increased cell proliferation is common. The MRN may present as polyclonal or monoclonal lesions at molecular analysis and with various degrees of atypia/dysplasia (17,18). Although they generally appear hypoechoic on ultrasonography, a hyperechoic aspect may be observed due to diffuse fatty change (19,20). The hyperechoic pattern is more frequent in high-grade dysplastic nodules (HGDNs) (19). Finally, the lack of a florid neoangiogenesis process makes most of these lesions hypovascular (21,22).


Figure 2
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  		Fig. 2. HCC can develop inside MRNs. This type of carcinogenesis is called nodule in nodule. However, most HCCs develop outside MRNs, and precancerous lesions can be identified in areas of large and small cell dysplasia or irregular regeneration (de novo carcinogenesis). (The histological images, stained with hematoxylin–eosin, have been kindly provided by Prof. W.F.Grigioni and Prof. A.D'Errico, Alma Mater Studiorum,University of Bologna).

 
According to the degree of cellular and structural atypia, MRN can be classified as large regenerative nodules without dysplasia, low-grade dysplastic nodules and HGDNs (17). The risk of progression to HCC increases along with the degree of dysplasia. During a mean follow-up of 33 months, Borzio et al. (20) observed a transformation rate toward HCC in 22% of large regenerative nodule, 25% of low-grade dysplastic nodule and 63% of HGDN. Nonetheless, 15% of the MRN disappeared during follow-up (87% of whom were large regenerative nodule/low-grade dysplastic nodule). Another feature suggesting the commitment to neoplastic transformation is monoclonality (18).

HGDN may be difficult to distinguish from well-differentiated HCC, particularly from the so-called ‘indistinctly nodular’ type of HCC (see below). Japanese pathologists view ‘stromal invasion’, i.e. hepatocyte invasion of portal tracts inside the nodules, as the hallmark of HCC (23).

However, most HCCs develop outside MRNs, and precancerous lesions can be identified in areas of large and small cell dysplasia (24) or irregular regeneration (25) (de novo carcinogenesis) (Figure 2).

Question 4: what are the morphological features of HCC at the early phases of its detectability, and how do they change over time?
Nodularity and growth type.
HCC usually arises as a single nodule in most patients with chronic liver diseases. Japanese authors have subclassified minute HCCs (up to 2 cm in diameter) as ‘indistinctly’ and ‘distinctly’ nodular due to this macroscopic feature (16). Indeed, indistinctly nodular HCCs are well-detectable nodular lesions on ultrasonography but appear rather indistinct at surgical examination, their texture being similar to the surrounding cirrhotic tissue. Histologically, they are very well-differentiated tumors containing portal tracts and bile ducts, with a ‘replacing’ type of growth and a scarce arterial vasculature leading to a hypovascular imaging pattern. By contrast, distinctly nodular HCCs are usually easily identifiable even at surgical inspection. Pressure on the surrounding liver caused by an ‘expansive’ type of growth of the tumor promotes collagen deposition in the stromal reticulum and its thickening, leading to the formation of a fibrous pseudocapsule around the tumor. These nodules are moderately differentiated, hypervascular and frequently disclose a microvascular portal invasion (up to 22%) (16). Since the histological features of indistinctly nodular HCCs suggest a lower degree of malignancy, a better course may be anticipated. Although a comparison of the natural history of these two types of tiny HCCs is unavailable, this assumption is supported by their outcome after curative resection. The ‘less malignant’ nodules recur later, less often, never locally and with fewer multiple recurrences, so that the 5-year rates of both overall survival (93%) and recurrence-free survival (47%) are much better than those of patients bearing minute but ‘overtly malignant’ HCCs (54 and 16%, respectively) (26). Therefore, the indistinctly nodular HCC marks a very initial stage of carcinogenesis as far as both the index lesion and the surrounding liver are concerned.

As the tumor further enlarges, it can maintain an expansive growth or assume an infiltrative pattern characterized by the wedging of malignant cells into sinusoids and cellular trabecula of the surrounding tissue, making the tumor boundary ill defined.

More than one-third of HCCs appear as multiple nodules (5,27). Multinodularity may result from either intrahepatic metastases from a primary focus or the occurrence of synchronous tumors. Synchronous tumors occur more frequently in chronic HBV infection, accounting for half of the multinodular cases (28). In a few patients (~5%), HCC appears as an infiltrative tumor (5). Again, HBV infection (alone or in combination with HCV infection) seems to be a risk factor for the development of infiltrative HCC (29).

Grade of differentiation.
The grade of differentiation tends to be correlated with the tumor size in HCCs characterized by expansive growth (30). Although this phenomenon cannot be considered a hard and fast rule (see previous paragraph), tumors <2 cm are generally well differentiated and, over time, the original neoplastic tissue is replaced by moderately to poorly differentiated cell clones because of the sequential accumulation of genomic mutations.

Vasculature.
Along with its growth and loss of differentiation, HCC blood supply becomes more and more dependent on newly formed arterial vessels (tumoral neoangiogenesis) and proportionally less dependent on the portal contribution. In parallel, sinusoid-like blood spaces go through a process of capillarization. This progressive imbalance between arterial and portal blood supply is responsible for the hypervascular pattern that characterizes HCC using enhanced imaging techniques (Figure 3) and represents the rationale for the use of transarterial chemoembolization in clinical practice and antiangiogenetic agents in experimental studies to control tumor growth (31).


Figure 3
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  		Fig. 3. (A) The grade of differentiation tends to be correlated with the tumor size in HCCs characterized by expansive growth. Along with its growth and loss of differentiation, HCC blood supply becomes more and more dependent on newly formed arterial vessels (tumoral neoangiogenesis) and proportionally less dependent on portal contribution. (B) The progressive imbalance between arterial and portal blood supply is responsible for the pattern characterizing HCC at enhanced imaging techniques: hypervascular in the arterial phase and hypovascular in the portal phase. Typical images of monofocal HCC at contrast enhanced ultrasonography (top) and computed tomography scan (bottom) are reported.

 
Microvascular invasion.
The risk of microvascular invasion in HCC with expansive growth increases with tumor size and dedifferentiation. However, among tumors ≤2 cm microvascular invasion is detectable in 20% of cases (32,33). Considering the indistinctly and distinctly nodular HCC <2 cm, portal microinvasion is found in 2 and 22%, respectively (16).

Question 5: how fast do tumors grow?
The mean volume doubling time of small (<5 cm) HCCs ranges from 112 to 204 days; the interindividual variability of tumor growth is also very high, the individual doubling time ranging from 30 to 600 days (34–38). The tumor growth may be linear (constant over time) or hyperbolic (initially slow then increasing) or parabolic (declining over time) (37).

To describe the risk of HCC progression in patients listed for liver transplantation, Cheng et al. (39) assumed that small HCCs may have either a Gompertzian type of growth, in which the initial exponential growth decreases as tumor size increases, or a rapid exponential growth. According to the Gompertzian model and a mean volume doubling time of 204 ± 132 days, a tumor 1 cm in diameter takes ~150 months (confidence interval 95%: 53–248) to grow beyond 5 cm of diameter. According to the model of rapid exponential growth, the same nodule takes a median time of only 48 months (confidence interval 95%: 17–79) to grow beyond 5 cm. By expanding these figures into the prediagnostic phase (Figure 1), some HCCs would take years to reach dimensions allowing their detectability (1–2 cm), whereas others can appear a few months after a previous negative imaging procedure.

Question 6: what are the factors predicting tumor growth and aggressiveness?
Both tumoral and extratumoral factors determine the growth rate and biological aggressiveness of HCC. Although not invariably (37), faster growth rate signifies a worse prognosis and tendency to relapse after surgical resection (36,40).

Tumoral factors.
Grade of differentiation and histological type.
The universally accepted criteria to classify HCCs, encompassing four degrees of cellular dysplasia and architectural tissue disarrangement, were proposed by Edmondson et al. in 1954 (41). There is clear evidence that the less differentiated the tumor is, the faster its growth and the higher the risk of vascular invasion and metastases (32,37,42). The trabecular type HCC is associated with longer volume doubling time (37).

Growth type.
With an expanding growth, the pressure of tumoral mass on the surrounding liver promotes the formation of a fibrous pseudocapsule. If the capsule is either absent or discontinuous, tumor cells may spread among non-tumoral liver cells and along sinusoids (infiltrative growth). A peritumoral capsule heralds a better prognosis after hepatic resection (43).

Neoangiogenesis.
Tumoral neoangiogenesis is a key process and appears to be a marker of aggressive tumor growth. Microvessel density predicts relapse after tumor resection (44) and biomolecular indicators of neoangiogenesis also have a prognostic meaning (see below).

Serum alfa-fetoprotein.
Alfa-fetoprotein (AFP) is a fetal glycoprotein whose circulating level quickly decreases after birth to <10 ng/ml. About 30–70% of HCCs produce AFP causing an elevation in plasma levels; in ~50% of these cases, AFP levels are directly proportional to cancer size (35). Moreover, AFP levels tend to parallel the tumor volume doubling time (34). An elevated AFP is an established predictor of recurrence after resection (45) and reflects a poor prognosis (5,46). A fucosylated variant of AFP, the so-called Lens culinaris agglutinin A-reactive AFP, correlates with cancer infiltrative growth, vascular invasion, low-grade differentiation, multiple cancer recurrence and poor prognosis (47,48). Although Lens culinaris agglutinin A-reactive AFP is assumed to be a more specific marker of HCC than total AFP, its advantage in clinical practice awaits further confirmation.

Biomolecular markers.
Following the advances in molecular biology, increasing efforts have been made to identify biomolecular predictors of tumor aggressiveness, as recently revised by Mann et al. (49).

Enhanced expression of ‘markers of proliferation’, such as proliferating cell nuclear antigen, Ki67, and ‘positive cell-cycle regulators’, including a variety of cyclins and cyclin-dependent kinases, have been found to predict relapse of HCC after resection and influence patients’ survival (49,50). Similarly, reduced expression of ‘cell-cycle inhibitors’, such as p27, is an independent predictor of poor prognosis (49).

Mutation of ‘proapoptotic’ genes, such as p53, is associated with a decreased survival (49). Enhanced activity of ‘apoptosis inhibitors’, such as BCL-xL, survivin and the recently identified PTMA and SET (which are involved in the caspase-dependent and caspase-independent apoptotic pathways, respectively), is related to HCC progression and indicate a poor prognosis (49,50).

Enhanced ‘telomerase activity’ predicts recurrence after hepatic resection (49). Among the ‘adhesion molecules’ studied, reduced expression of E-caderin predicts a poor prognosis, whereas the prognostic value of β-catenin expression and its cellular localization are controversial (49).

Alterations of ‘hormonal receptors’, ‘growth factors and related receptors’ may also be predictors of cancer aggressiveness. Namely, activating mutations of estrogen receptors are associated with poor prognosis (51), as well as the overexpression of the growth factors c-myc, ras and hepatocyte growth factor or the receptors c-met and the epidermal growth factor receptor family (49). Moreover, an overexpression of leptin receptors would predict a better survival (49), and high levels of placenta growth factor would herald an early recurrence of HCC after resection (52).

A variety of ‘matrix metalloproteinases’ and the ‘plasminogen activator system’ is involved in the process of metastasis and appear to have prognostic value in HCC (49).

Another category of prognostic determinants is that of the angiogenetic regulators. Serum and/or tissue levels of vascular endothelial growth factor are directly proportional to microvascular density and correlate with the degree of cellular dedifferentiation (49). They would herald tumor relapse with a poor prognosis, but this association has not been confirmed by other studies (49). Enhanced expression of hypoxia-inducible factor1a, the master regulator of hypoxia-induced gene expression, promoting neoangiogenesis, has been found in tumors with poor prognosis and high recurrence rate after resection (49,50).

More recently, DNA microarray techniques, which analyze the simultaneous expression of multiple genes, have been used in an attempt to identify survival subclasses; the low survival subclass included tumors with a strong expression of cell proliferation and antiapoptosis genes, higher expression of genes involved in ubiquitination (probably responsible for selective degradation of critical proteins including cell-cycle inhibitors), enhanced expression of histone variants (involved in chromosome breaks response) and higher expression of hypoxia-inducible factor1a (50). Finally, a new subtype of HCC that shares a gene expression pattern with fetal hepatoblasts has been recently identified (53). This subtype probably arises from adult hepatic progenitor cells (hepatoblast subtype) and has a poorer prognosis compared with the hepatocyte subtype, which would derive from mature hepatocytes (Figure 4). Differential expression of genes involved in invasion and metastasis may account for the different prognosis in the two subtypes.


Figure 4
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  		Fig. 4. Shared gene expression patterns between some HCCs and fetal hepatoblasts suggest that a subtype of HCC (hepatoblast subtype) may arise from bipotential adult hepatic progenitors cells, whereas the majority of human HCCs (hepatocyte subtype) would derive from mature hepatocytes. However, the expression profile of the hepatoblast HCC could also result from a dedifferentiation process of transformed mature hepatocytes. Individuals with the hepatoblast subtype have a worse prognosis than those with the hepatocyte subtype.

 
The number of ‘genetic and karyotypic alterations’ is inversely related to the degree of differentiation (13,49,54), and several authors have observed a relation between poor prognosis and chromosomal losses or gains. Chromosomal loss on 17p13.3 and gain on 8q11 as well as the total number of genetic alterations and loss of 13q are independent predictors of poor prognosis (49). Laurent-Puig et al. (55) reported that chromosomal instability and mutation in p53 were related to HBV infection, poorly differentiated tumors, and chromosome losses on 9p and 6q were specifically associated with a poor prognosis. On the other hand, chromosomal stability and β-catenin-activating mutations were associated with 8p loss of heterozigosity and non-HBV large-size HCCs (55). Other authors report more frequent β-catenin mutations in well-differentiated HCCs (49).

Extratumoral factors.
Immune response.
As for other tumors, the natural history of HCC is influenced by the host immune response. For example, the immune suppression of transplanted patients is associated with a very aggressive clinical course and high tendency to metastasize of recurrent or de novo tumors (56). Similarly, in patients with HIV infection, HCC more frequently exhibits a multinodular pattern with infiltrative growth and seems to have a worse prognosis (57). Moreover, the density of lymphocyte infiltration in the nodule (58), a surrogate marker of the antitumoral immune response, and the activation of the host natural killer cells (59) are inversely correlated with biological aggressiveness and the risk of recurrence after resection of the tumor. Finally, the relative predominance of negative immune modulator CD4+/CD25+ lymphocytes in the blood or peritumoral tissue seems to be associated with both the risk and rapid progression of HCC (60).

Sex.
Male sex is an established risk factor for HCC in patients with chronic liver diseases (61), whereas gender influence on HCC progression and prognosis remains controversial. Some studies (5,62), but not others (63,64), report a worse prognosis in males. Androgen hormones have been claimed as responsible for the greater cancer risk and grim prognosis (61).

Age.
Although advanced age resulted as a negative prognostic factor in some patient series (65,66), there is no evidence that advanced age per se is associated with a greater cancer aggressiveness. Confounding factors such as shorter life expectancy, higher prevalence of comorbidities and less ‘aggressive’ therapeutic management could explain the negative impact of age on prognosis. In fact, the adverse effect of age disappeared when patients were segregated according to the treatment received (65,66).

Viral etiology.
HBV infection has been associated with a worse prognosis, especially in patients bearing the estrogen receptor mutation and patients with advanced tumors (67,68). A greater aggressiveness of these tumors is thought to derive from the high chromosomal instability caused by HBV (28).

Question 7: what is the presentation of HCC and its evolution in the clinical phase?
The clinical presentation of HCC is mainly determined by the degree of liver dysfunction and the tumor burden. The latter greatly depends on the ‘timing’ of diagnosis. Differences involving these factors can largely explain the geographical variability in clinical presentation, whereas the ethnic diversity is probably of secondary importance.

In Western countries and Japan, with implementation of surveillance programs of high-risk patients, HCC is frequently diagnosed at an early asymptomatic stage. In Italy, 45% of HCCs occurring in the setting of chronic liver diseases are diagnosed under surveillance and in the preclinical phase (5). More than 60% of HCCs diagnosed under surveillance are non-advanced cancer, whereas this percentage drops to about one-third for the HCCs detected outside surveillance (5). The most common presenting symptoms and signs are hepatomegaly, abdominal pain/discomfort, jaundice, ascites and constitutional syndrome (fatigue, malaise, fever and weight loss) (42). Less commonly, digestive or peritoneal bleeding and hepatic encephalopathy reveal the presence of HCC.

On the contrary, HCCs arising in a non-cirrhotic liver usually present as large solitary symptomatic masses, detected outside surveillance programs. They exhibit a fast and infiltrative growth with frequent vascular invasion and metastases (42). These clinical features are probably due to delayed diagnosis since these tumors, as compared with HCCs occurring on cirrhotic livers, present a similar or lower grade of cell dedifferentiation, a higher prevalence of peritumoral capsule formation and an equally or less frequent vascular invasion (42,69).

Size and number of tumoral nodules, invasion of the main vessels, metastases, general performance status, AFP value and liver function are the most important clinical variables that have been combined to create prognostic systems and to drive therapeutic decisions (70). However, the prognosis of ‘untreated’ HCC, which is the best representation of the natural history, is still largely undefined. Considering the ‘early’ HCC, a study including 22 Eastern cirrhotic patients (a third with advanced cirrhosis) bearing a tumor <3 cm in diameter, reported survival rates of 91, 55 and 13% at 1, 2 and 3 years, respectively (35). The corresponding survival rates in 39 Italian patients with HCC <5 cm (multifocal in 15 cases) were 81, 56 and 21% (37). After stratifying patients according to the stage of cirrhosis, the 2 year survival rate attained 82% in Child-Pugh A patients and was only 36% in Child-Pugh B–C cases.

Considering ‘advanced’ HCC, in two studies including Caucasian patients with unresectable tumors, survival rates at 1, 2, 3 and 5 years were 54–72, 40–41, 28–38 and 20%, respectively (51,63). Survival was much better in the absence of systemic symptoms, vascular invasion or metastases with respect to patients with at least one of the three variables (at 3 years: 50 versus 8%) (63).

Although HCC may show an early microvascular invasion and tend to infiltrate large vessels over time, extrahepatic metastases generally appear in the late stage. Indeed, although about one-third of patients with unresectable HCC present vein invasion at 3 years after diagnosis, only 22% develop metastases over this period (63). The most common sites of metastases are lungs, followed by local lymph nodes, bone and adrenal glands (42,69).

The most frequent clinical features of tumor progression include ascites, hepatic encephalopathy, bacterial infections and digestive bleeding. In addition, more than half of patients with unresectable HCC and without pain at diagnosis will develop these symptoms in the following 3 years (63).

In the ‘terminal stage’, characterized by a tumoral replacement exceeding 50% of the liver mass, vascular invasion and systemic symptoms, survival ranges from several weeks to a few months (46,63,71).

Question 8: what are the causes of death in HCC patients?
Cancer progression is the most frequent cause of death in HCC patients, both in those without chronic liver diseases, in whom it accounts for virtually all cases, and in cirrhotic patients (~60% of deaths). Other causes of death include liver failure (7–30%), digestive bleeding (7–10%), infections (2%) and pulmonary embolism (1%) (5,63,72). Intraperitoneal bleeding due to tumor rupture is uncommon in Western countries.

Question 9: has the mortality rate for HCC changed in recent years?
A large retrospective population-based study, including >7000 unselected USA patients, showed that the 5 year survival rate remains very poor (2% in the period 1977–1981 and 5% in 1992–1996) (73). Similarly, in the different European regions, these figures were 0.9–4.9% in the early 1980s and 4.6–7.9% in the mid-1990s (74). An advanced cancer stage at diagnosis and/or the frequent coexistence of advanced cirrhosis precluding curative or effective treatments in many patients are the reasons for such dismal figures. However, data from population-based studies are much worse than those reported in clinical investigations derived from referral centers. The discrepancy can be explained not only by the selection bias affecting clinical studies (including more patients with non-advanced tumors) but also by the gap still separating the state-of-the-art management of HCC—which is extremely complex—from the everyday practice, where many patients are ‘undertreated’ or ‘overtreated’ (73).

Finally, there is evidence to suggest that the continuous refinement of HCC treatment has a favorable impact even in centers dedicated to this disease. A recent prospective study including 417 patients with compensated cirrhosis maintained on regular surveillance demonstrated a sharp decline in the mortality over 15 years, mainly because of the decrease in mortality of the treated patients (72). Therefore, a general improvement in HCC prognosis is expected to occur in the near future, due to the increasingly widespread implementation of surveillance programs for high-risk patients and the application of patient-tailored treatment (70).

Question 10: do the advances in the knowledge of the natural history of HCC offer new perspectives for the management of HCC patients?
None of the human tumors has its natural history influenced by the interaction of so many factors as HCC. In fact, such an interplay not only involves the cancer cell biology and the host's innate and specific immune response but also different etiologies (hepatotropic viruses, toxic agents and genetic conditions) and the very frequent coexistence of liver cirrhosis. This unique condition accounts for the huge heterogeneity of molecular pathogenesis and biological aggressiveness of HCC as well as the protean epidemiological and clinical features of this neoplasm, such as the existence of well identifiable risk populations and precancerous conditions, its stringent dependence on the development of cirrhosis, its variable period of clinical silence, its non-specific presentation and the need for treatments tailored on both tumor burden and severity of cirrhosis.

We think that our ‘Question–Answer’-based review has plainly stated the case for an updated knowledge of the natural history of HCC in order to decipher the heterogeneous and ever-changing clinical features of this cancer and to refine its management. Indeed, a better understanding of the natural history achieved in the last two decades has promoted practices able to improve the prognosis of HCC patients. These include the implementation of surveillance programs of risk patients aimed at diagnosing HCC at an early stage suitable for radical treatments, a selection of the most appropriate treatment according to both tumor stage and hepatic function and a search for agents selectively blocking growth factors or vital ‘molecular pathways’ of cancer cells and/or their vascular supply.

In the near future, further advances in the comprehension of pathogenesis and natural history of HCC—and the consequent positive effects on its management—are expected from genomic and proteomic research. Indeed, this is a very promising route to follow, as these modern approaches may: (i) identify efficient tissue and serum markers for the early diagnosis of the tumor; (ii) disclose gene signatures and molecular profiles predicting the biological behavior of both HCC and its histological precursors with much greater accuracy than that achievable at present; (iii) reveal specific molecular targets for the new pharmacological ‘bullets’ that are appearing in the armamentarium to battle against HCC.


   Acknowledgments
 
Conflict of Interest Statement: None declared.


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Received February 19, 2008; revised April 7, 2008; accepted April 30, 2008.


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