Biology of Hepatocellular Carcinoma
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- Pang, R.W.C., Joh, J.W., Johnson, P.J. et al. Ann Surg Oncol (2008) 15: 962. doi:10.1245/s10434-007-9730-z
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Hepatocellular carcinoma (HCC) is a common cancer in the world due to high prevalence of hepatitis B or C virus infection. Research in recent years has uncovered important molecular pathways involved in development and progression of HCC. Several genetic aberrations and molecular mechanisms responsible for initiation of hepatocarcinogenesis have been identified. Novel biomarkers for HCC are being developed for better detection and prognostication. Alpha-fetoprotein, the conventional marker of HCC, has limited sensitivity and specificity. Serum levels of isoforms of AFP based on differential lectin binding of the glycan moiety appear to be more sensitive and specific than total AFP level in early detection of HCC. The clinical usefulness of other HCC biomarkers such as des-γ-carboxy prothrombin and glypican-3 are under investigation. HCC is an aggressive tumor with early vascular invasion and metastasis. Studies over the past two decades have elucidated the clinical predictors of outcome, leading to several staging systems for HCC based on clinical parameters. However, the predictive accuracy of clinical staging systems is limited. Recent studies suggested that biological factors may provide additional prognostic information. In particular, gene expression profiling appears to be a promising approach. Study of tumor angiogenesis in HCC reveals that the expression of angiogenic factors such as vascular endothelial growth factor and angiopoietins may also predict prognosis. The elucidation of tumor biology of HCC is of particular importance in the current era of rapid development of anti-cancer molecular targeting agents, which provide hope for an effective systemic therapy for HCC.
KeywordsHepatocellularBiomarkersBiological factorsAngiogenesisMolecularGene expression
In recent years, the importance of understanding the molecular biology of HCC has gained wider emphasis as molecular targeting therapy has shown to be effective in other cancers. More data are now available on the molecular pathways in HCC, and several molecular targeting agents have shown promise for the treatment of HCC in preclinical studies.4 In this article, the molecular mechanisms of HCC carcinogenesis, the development of novel biomarkers, the clinical and molecular predictors of outcome, and the significance of tumor angiogenesis in HCC are reviewed.
Molecular Mechanisms Of HCC Carcinogenesis
Chronic HBV infection is the most common etiology of HCC in Asian countries. HBV is a DNA virus, sharing with oncogenic retroviruses a unique replication strategy through reverse transcription and a characteristic life cycle that includes integration into the host genome. Integration of the HBV viral DNA in human genome can induce chromosomal instability and results in rearrangements or deletions.5 Insertional mutations resulting from genome integration of HBV viral DNA at specific sites can activate endogenous genes such as cyclin A and SERCA1, which play important roles in regulation of cell division or viability, culminating in uncontrolled cell proliferation.6,7 Integration of HBV DNA may also lead to deregulated expression of certain oncogenes or tumor suppressor genes that control cell survival or cell death.8
Another mechanism by which HBV induces hepatocarcinogenesis is through the expression of viral proteins that have oncogenic property, in particular the X protein (HBx).9 HBx activates different promoter elements and trigger activation of transcription factors like AP-1 (activator protein-1) and NF-kB (nuclear factor kappa B).10,11 Through its transactivation activity, HBx affects the expression of a variety of genes that are involved in the control of the cell cycle, proliferation or apoptosis. One important tumor suppressor gene p53 is affected by HBx. HBx protein has been shown to repress p53-mediated transcriptional activation and inactivates p53 through direct binding, thus inactivating p53-dependent activities including p53-mediated apoptosis.12 The modulatory effects of HBx on p53 provide a possible mechanism of malignant transformation of HBV infected liver cells. The transactivation activity of HBx is in turn affected by the expression of other genes. For example, it has been demonstrated that the transactivation activity of HBx is synergistically enhanced by a peptidyl prolyl isomerase named PIN1, which is overexpressed in HCC and is a regulator of β-catenin signaling and cyclin D1 regulation.13,14 In addition to the interaction of HBx with the transcription machinery, there is evidence that HBx interferes at multiple steps with DNA repair and so may confer an increase of critical mutations.15
Alfatoxin, a mycotoxin from Aspergillus flavus and Aspergillus parasiticus, is a carcinogen of HCC commonly found in Asia and sub-Saharan Africa, where the molds grow in nuts and grains improperly stored under hot and humid conditions. These areas also have a high prevalence of HBV infection. Although aflatoxin alone is a significant etiological factor for the development of HCC, in regions in which exposure to aflatoxin is high and HBV infection is prevalent, these two factors act synergistically in hepatocarcinogenesis by leading to common mutations.16 In order for aflatoxin (AFB1) to damage hepatocyte DNA, it must be converted to its exo-8,9-epoxide by hepatic cytochrome p450.17 After conversion, AFB1-8,9-epoxide reacts with guanine nucleotides in the hepatocyte DNA to form a number of adducts, which consequently lead to genetic changes that contribute to hepatocarcinogenesis. Of significance, aflatoxin can induce mutations of the p53 gene, which appears to be a significant molecular event in malignant transformation in some HCC.18,19 There is evidence that p53 mutation may be a common genetic event induced by both aflatoxin and HBV, and this partly accounts for their synergism in hepatocarcinogenesis.20,21
Unlike HBV, HCV is an RNA virus that does not integrate into the host genome. HCV induces hepatocarcinogenesis via host—viral protein interactions. The HCV proteins that have demonstrated to be associated with hepatocarcinogenesis include core, NS3, and NS5A proteins, which have all been shown to inhibit post-transcriptional expression of p21,WAF1 a cyclin-dependent kinase inhibitor that plays a major role in cell cycle control.22–24 The HCV core protein seems to play a particularly important role in HCV hepatocarcinogenesis through modulation of cellular proliferation, apoptosis, and immunological responses. The HCV core protein has transcriptional regulating functions on different cellular genes involved in regulation of cell growth, including the proto-oncogene c-myc.25 Transgenic mice expressing the HCV core protein develop hepatic tumors that first appear as adenomas, within which then evolves a HCC, presenting the nodule-in-nodule histopathological feature typical of early HCC patients with chronic HCV infection.26 HCV core protein may also induce carcinogenesis through other mechanisms such as inhibition of multiple activators of apoptosis, including both Fas activation and tumor necrosis factor-α.27
Development Of Novel Biomarkers Of HCC
Tumor markers of hepatocellular carcinoma
Established tumor markers
Lens culinaris agglutinin-reactive AFP (AFP-L3)
Potential novel tumor markers
Recent effort has been directed towards the search for a type of AFP that is specific for HCC, i.e., distinguishable from the AFP that arises in chronic liver disease. The answer for a specific AFP appears to lie in the glycan moiety of the AFP, and clinically applicable diagnostic assays based on differential lectin binding of the glycan moiety have been developed.34,35 The various glycoforms of AFP have been characterized, showing that there is indeed a clear difference based on the degree of sialylation; AFP arising from germ cell tumors, HCCs and cirrhosis appears distinct.36,37 Such isoforms can be detected before clinical evidence of the disease arises.38 An assay that can detect these changes has been developed but it is not currently available in a state that is readily transferable to routine laboratories.39 Lens culinaris agglutinin (LCA)-reactive AFP (also named AFP-L3) is an isoform of AFP that has been shown to be very specific for HCC and may improve the detection of small HCC.40 Furthermore, AFP-L3 as a marker for HCC is superior to the total AFP not only in the diagnosis of HCC, but also in predicting the prognosis of the patients.41
Another tumor marker of HCC that has been used in some centers is des-γ-carboxy prothrombin (also known as protein induced by vitamin K absence or antagonist-II, PIVKA-II). Production of PIVKA-II is the result of an acquired defect in the post-translational carboxylation of the prothrombin precursor in HCC cells.42 PIVKA-II has a higher specificity for HCC than AFP but is less sensitive. Serum PIVKA-II level is found to be elevated in around 50–60% of patients with HCC but only in 15–30% of patients with HCC <3 cm.43,44 Serum PIVKA-II and total AFP levels are independent of each other in HCC patients, and some studies have shown that combined use of AFP and PIVKA-II increases the sensitivity as well as the specificity for diagnosing HCC.45,46 Serum PIVKA-II level could also be used as a prognostic indicator for HCC patients, and may be more useful than AFP in reflecting the invasive characteristics of HCC.47,48
Glypican-3 (GPC3) is a novel tumor marker of HCC that has been widely studied recently. It is a heparan sulfate proteoglycan anchored to the plasma membrane and is capable of modulating the activities of several growth factors.49 GPC3 expression has been demonstrated to be increased in HCC, and GPC3 can be detected in serum in 40–53% of HCC patients, whereas it is not detectable in the serum of healthy individuals.50,51 There is no correlation between serum GPC3 and AFP levels in patients with HCC. It has been shown that combination of GPC3 and AFP increases the sensitivity without affecting the specificity in diagnosis of HCC.51 Furthermore, soluble GPC3 (sGPC3), the NH2-terminal portion of GPC3, seems to be superior to AFP in the sensitivity of detecting well or moderately differentiated HCC, and the simultaneous determination of both markers improves overall sensitivity from 50% to 72% in one study.52 However, more studies are required to determine the usefulness of this new marker in early diagnosis of HCC.
Potential novel tumor markers for HCC are being identified using modern high-throughput genomic or proteomic approaches. For example, through cDNA microarray study of HCC tissues, a novel growth factor granulin-epithelin precursor (GEP) has been found to be overexpressed in HCC and detectable in serum of HCC patients, suggesting that it may be a potential novel tumor marker for HCC.53 Using the surface-enhanced laser desorption/ionization (SELDI) time-of-flight (TOF) mass spectrometry (MS) approach, proteomic profiles for both HBV and HCV related-HCC have been studied, and serial changes consequent on tumor resection may allow identification of specific tumor markers for HCC patients.54–56 Many of the peptide or proteins that make up the characteristic SELDI profile can now be identified and, on the basis of experience with other tumor types, the possibility that a convenient chip-based multimarker assay can be developed is close to reality.
Clinical Predictors Of Outcome
Tumor factors predictive of prognosis
Large tumor size
Macroscopic vascular invasion
Microscopic vascular invasion
Microscopic satellite nodules
High serum alpha fetoprotein level
Liver fibrosis / cirrhosis
Tumor size is considered to be a significant risk factor for intrahepatic and extrahepatic spread of HCC, and large tumor size has been associated with decreased disease-free and overall survival.61–63 The Liver Cancer Study Group of Japan reported that tumor size and survival after liver resection for HCC were inversely correlated.63 Virtually all major HCC staging systems such as the Cancer of the Liver Italian Program (CLIP) staging,64 the Barcelona Clinic of Liver Cancer (BCLC) staging,65 and the American Joint Committee on Cancer (AJCC) tumor–node–metastasis (TNM) staging66 include tumor size as a prognostic factor, albeit with varying discrete cut-off values. While patients with large tumors have a worse prognosis after resection or liver transplantation in general,59,60,67 several investigators have reported series of patients with large HCC (>5 to 10 cm) treated surgically with 5-year survival in the range of 25% to 39%.68–70 Tumor size predicts vascular invasion and histological grade, two of the most potent factors associated with HCC outcome.71–73 The fact that tumor size is often a marker for vascular invasiveness and tumor grade may explain why tumor size often fails to affect survival in studies that control for these other factors.74 In fact, long-term survival can be expected in patients with solitary tumors regardless of size in patients with no underlying fibrosis or vascular invasion. As such, patients with large HCC who have an otherwise resectable lesion should not be excluded from surgical consideration.
Multiplicity of tumors is also an adverse prognostic factor included in several HCC staging systems including CLIP, BCLC, and AJCC. Multiple tumors arise from either intrahepatic metastases from one dominant tumor or multicentric de novo tumors. Although patients with multiple lesions in general have a worse prognosis than patients with solitary HCC, resection of multinodular disease (more than three lesions) can be associated with 5-year survival of 40%.75 In addition, the prognosis of patients with multiple tumors, none larger than 5 cm, is similar to that of patients with a single tumor with vascular invasion.76
Although microscopic vascular invasion cannot be clinically detected in the preoperative setting, HCC invading major portal and/or hepatic veins can be identified by modern imaging. Several studies have reported that tumor size and multiplicity of tumors did not affect prognosis in patients with major vascular invasion.63,77,78 These results suggest that major vascular involvement may be the most important prognostic factor in patients with HCC. A 5-year survival of 7–10% has been reported for HCC patients with major vascular invasion treated by hepatic resection.77,78 The presence of microscopic venous invasion in histopathological examination is the most consistently reported risk factor of postoperative recurrence after resection or liver transplantation for HCC.69,73,74,79 It is widely accepted that microscopic metastasis via portal venous system is an important mechanism for intrahepatic recurrence after resection of HCC. The presence of microscopic satellite nodules is another indicator of tumor invasiveness.79
Although not as universally accepted as other clinical prognostic factors, high serum AFP level has been reported to be associated with worse survival after surgical or nonsurgical treatment of HCC.28,29,69 Some staging systems such as the CLIP staging classification scheme include AFP as a prognostic factor. An elevated serum AFP level may be indicative of a large tumor burden and more extensive disease, and thus a worse prognosis. Some authors have even suggested that high serum AFP levels may suppress the ability of the immune system to destroy cancer cells.80,80
In addition to tumor factors, host factors may also affect the prognosis of the HCC patients. Previous studies have noted that underlying hepatic fibrosis/cirrhosis had an adverse effect on long-term survival after resection of HCC.82,83 Underlying liver fibrosis is associated with worse liver function that may reduce the chance of long-term survival. Liver fibrosis may also indicate pathological and genetic changes throughout the liver leading to a field cancerization effect, which increases the risk of multicentric hepatocarcinogenesis. The most recent AJCC staging edition accounts for a separate reporting of fibrosis in every resected case of HCC.66 Studies correlating serologic evidence of hepatitis B virus (HBV) or hepatitis C virus (HCV) infection with clinicopathological features and prognosis in patients with HCC have been inconsistent. Yamanaka et al.84 reported that HCC patients with HCV had a worse 5-year survival (42%) compared with HBV patients (54%). In contrast, Haratake et al.85[T1] noted that HCC patients with HCV had an improved survival compared with patients with HBV. Pawlik et al.86 reported that hepatitis serology predicted tumor and liver-disease characteristics but not prognosis after resection of HCC. Based on the current data, viral hepatitis status does not appear to be a strong predictor of prognosis following resection of HCC. As such, the treatment for HCC should be dictated by the extent of underlying liver disease rather than by hepatitis viral serology.
Biological Predictors Of Outcome
Prognostication based on clinicopathological factors can only predict approximate outcome as these factors merely reflect the likely biological behavior of the tumor based on morphological and histological features. The AJCC TNM staging, which encompasses multiple tumor factors including tumor size, tumor multiplicity, vascular invasion, lymph node metastasis, and distant metastasis, has been the most prevalent method of estimating the early prognosis of HCC patients after surgery. However, it is not yet accepted as the definitive staging system for HCC because it cannot reliably predict outcome in all HCC cases. This uncertainty inherent in the conventional staging systems may be attributed to incomplete exploration of the biological differences of tumors in patients belonging to identical classification subgroups. As tumor phenotype likely reflects underlying genetic alterations, direct molecular study of these alterations may be beneficial in understanding not only the morphological features but also the biological behavior of tumors.87
Several tumor biological factors such as DNA aneuploidy,88 high tumor cell proliferation index,89 high telomerase activity,90 and mutation of the p53 gene91 have been associated with increased risk of postoperative tumor recurrence in HCC. However, the prognostic impact of these factors remains uncertain because of limited number of studies and conflicting data in some studies.79 Recently, there has been great interest in the study of gene expression profile in relation to prognosis of HCC patients. The cDNA microarray is a modern technology that allows analysis of thousands of genes simultaneously using gene chips. Molecular-based prediction of tumor recurrence may allow clinicians to make better decision about the use of adjuvant therapy which might improve patients’ prognosis. Several studies have identified gene expression profiles predictive of the recurrence or prognosis of HCC patients.92–95 Kurokawa et al.95 analyzed gene expression of tumors using a polymerase chain reaction (PCR)-based array of 3072 genes in 100 HCC patients treated by liver resection. In an estimation group of 60 patients, 92 genes demonstrated distinct expression patterns differing significantly between recurrence cases and recurrence-free cases. Using the 20 top-ranked genes with the highest prediction accuracy as a molecular signature of recurrence, early intrahepatic recurrence was correctly predicted for 29 of 40 cases in a validation group, and the 2-year recurrence rates in the patients with the good signature and those with the poor signature were 29.4% and 73.9%, respectively. Multivariate Cox analysis revealed that the molecular signature comprised of the 20 genes was an independent indicator for recurrence. In another study, the same group of authors analyzed the expression of 3,080 genes in 20 HCC patients who were treated with combination chemotherapy.96 The study showed that molecular analysis of 63 selected genes can predict the response of HCC patients with major portal vein tumor thrombi to combination chemotherapy with 5-fluorouracil and interferon-alpha, suggesting that gene expression profile study may predict outcome not only in surgical patients but also in patients with advanced HCC receiving nonsurgical treatment. These studies illustrate the potential of genomic technologies to advance treatment planning beyond the empiric, towards a more molecularly defined, individualized approach. The combination of a novel biological indicator and conventional clinical indicators of prognosis such as the TNM stage will more accurately predict the patients’ outcome, enabling more appropriate therapeutic decisions for HCC patients.97 However, the available studies in the literature are limited by sample size, and the gene signatures identified in different studies vary considerably. It is mandatory that the prognostic value of gene expression profiling be validated in large samples of HCC patients from different centers to further evaluate its potential use in clinical practice for prognostication.
In addition to its potential value in prognostic prediction, gene expression profiling has been used to delineate clonality of multifocal tumors by differentiating metastatic tumor nodules from multicentric tumor nodules, as tumors of similar clonality share similar gene expression profiles.98 Furthermore, cDNA microarrays also allow the identification of genes that are highly expressed in HCC that can be further characterized in order to uncover novel tumor markers, oncogenes, and therapeutic targets for HCC.99,100
Tumor Angiogenesis In HCC
VEGF promotes the growth, migration, and morphogenesis of vascular endothelial cells and increases vascular permeability.104 Increased VEGF expression has been shown in various human malignant tumors, including brain tumor, bladder cancer, ovarian cancer, colorectal cancer, gastric cancer, breast cancer, and HCC.105 Moreover, it has been reported that VEGF expression correlated with tumor angiogenesis, aggressive tumor behavior, and even with its prognosis in several malignant tumors. Pathological correlation studies have demonstrated that high VEGF expression in HCC is associated with a high proliferative index, poor encapsulation of tumors, and venous tumor emboli portal vein thrombosis.106,107 Furthermore, high serum VEGF levels have been shown to predict microscopic venous invasion and poor prognosis after resection of HCC.108,109 In HCC patients, high serum VEGF levels have also been shown to correlate with increased recruitment of endothelial progenitor cells from bone marrow, which is believed to contribute to tumor angiogenesis.110 However, the importance of VEGF expression and its relationship to clinicopathological features are still not entirely clear. Some investigators have shown that VEGF expression in HCC was not different from that in the corresponding nontumorous portion of the liver,111 while other investigators found that VEGF expression did not correlate with angiogenesis or any clinicopathological features of HCC, suggesting that angiogenesis of HCC is not controlled by VEGF alone but is also mediated by other angiogenic factors.112
Recently, angiopoietins (Ang) have been identified as a group of ligands of the tyrosine kinase Tie-2 receptor that play a role in regulating tumor angiogenesis.113 The angiopoietins include both receptor activators and antagonists. Binding of angiopoietin 1 (Ang-1) causes autophosphorylation of Tie-2, whereas binding of angiopoietin 2 (Ang-2) suppresses autophosphorylation of Tie-2. The Tie-2 signal is, therefore, defined by the quantitative balance of activities between Ang-1, the agonist, and Ang-2, the antagonist. It has been also reported that proper regulation of tyrosine kinase Tie-2 is absolutely required for normal vascular development, apparently by regulating vascular remodeling and maturation.113 The Ang-1/Tie-2 pathway is thought to mediate the vital functions of vascular stabilization and vascular remodeling, via integration of periendothelial cells into the vascular wall, particularly in the presence of VEGF. In contrast to Ang-1, Ang-2 induces vascular regression in the absence of VEGF but increases vascular sprouting in its presence. It has been shown that the ectopic expression of Ang-2 in HCC cells promotes rapid development of tumor and aggravates its prognosis, suggesting that the Ang-2/Tie-2 pathway might be involved in angiogenesis of HCC.114 In some clinical studies, Ang 2 expression was significantly related with tumor progression and patient’s prognosis. Patients with high Ang 2 mRNA expression by reverse transcription PCR had a poor prognosis and earlier tumor recurrence.114,115 Tie-2 expression has also been related with tumor progression in HCC, but its exact clinicopathological significance is unclear.116
Increased expression of VEGF and Ang-2/Tie-2 appears to play a critical role in promoting tumor angiogenesis in human HCC. Other angiogenic factors have also been shown to be involved in tumor angiogenesis or progression of HCC, including basic fibroblast growth factor,117 platelet-derived endothelial cell growth factor,118 tissue factor,119 cyclooxygense-2120 and angiogenin.121 However, the relative role of these angiogenic factors in HCC is unclear. The elucidation of the mechanisms of angiogenesis is of importance because anti-angiogenic agents are now available and may be of potential benefit in patients with HCC.122
HCC is one of the most aggressive cancers, associated with poor prognosis despite significant advances in surgical and locoregional therapies over the past two decades. Most patients present with advanced disease, and only a small proportion of patients are amenable to resection or liver transplantation. The development of novel biomarkers of HCC may help early detection of HCC and increases the chance of curative therapy. The prognosis of HCC patients remains unsatisfactory even with surgical treatment because of the high rate of recurrence. Current staging classifications based on clinical parameters are limited in prognostic accuracy. Study of biological factors and gene expression profiles may refine the prognostic classification of HCC.
The study of tumor biology is also important to develop novel agents for chemoprevention and treatment of HCC. Understanding the molecular mechanisms of hepatocarcinogenesis may lead to effective strategies in chemoprevention among patients who carry the risk factors such as hepatitis B or C viruses. HCC is well known to be resistant to conventional chemotherapy. The development of an effective systemic therapy for HCC is of paramount clinical importance. The recent data from studies on the molecular biology of HCC suggest that molecular targeting therapies aiming at inhibition of tumor angiogenesis, blocking of proliferation signaling pathways or triggering of apoptosis may provide novel effective therapies for HCC.