Introduction

epatocellular carcinoma (HCC) is one of the most common tumors in the world with an esti mated 500 000 to 1 000 000 new cases per year. Although less frequent in the United States and Europe, these tumors have an annual incidence of up to 500 cases per 100 000 population in certain regions of Asia and sub-Saharan Africa. The reasons for this high incidence are chronic infections with hepatitis B virus (HBV) or hepatitis C virus (HCV) as well as HBV-HCV coinfections.^[1-3] The clinical course of HBV and HCV infection depends partly on molecular characteristics of the viruses, on patient¡¯s HLA haplotype, and on other coexisting risk factors. Well recognized non-viral exogenous agents associated with the pathogenesis of HCC are alcohol and aflatoxins. In the west, alcohol induced liver injury is a leading cause of liver cirrhosis and the most important HCC risk factor. In southern China and Africa, dietary ingestion of high levels of aflatoxin B1 (AFB1) may represent a special environmental hazard, particularly in chronic HBV carriers.

The major clinical risk factor for HCC development is liver cirrhosis and 70%-90% of HCC develop in a macronodular cirrhosis. The HCC risk in patients with liver cirrhosis depends on the activity, duration and etiology of the underlying liver disease. In addition, HCC is more frequent in men than in women and the incidence generally increases with age also in the west.

The prognosis of patients with inoperable HCC is very poor, while patient¡¯s est

rogen receptor status is the strongest prognostic factor for survival.^[4] Despite progress in early diagnosis and surgical or nonsurgical strategies, the overall survival of HCC patients has not been significantly improved during the last two decades.^[5] Preventive strategies, therefore, are of paramount importance and need to be actively explored in order to reduce the incidence of HCC. In the following, gene therapeutic strategies as well as some targets for the prevention of HCC will be discussed.

Hepatocarcinogenesis

 Central to the concept of molecular carcinogenesis are mutations of oncogenes and tumor suppressor genes as well as genetic instability of cellular DNA, including mismatch repair deficiency and impaired chromosomal segregation. In hepatocarcinogenesis, these genetic events occur in the setting of liver cell injury and necrosis associated with an increased rate of hepatocyte regeneration and mitosis. Any exogenous agent, viral or other, that contributes to chronic low grade liver cell damage and mitosis potentially increases the risk of HCC development, rendering liver cell DNA susceptible to additional genetic alterations. Also, chronic immune mediated liver cell injury may trigger HCC development in the absence of viral transactivators, insertional mutagenesis and genotoxic chemicals.^[6] Overall, there are a variety of molecular and immunological mechanisms by which endogenous, environmental and viral factors may play an interactive role in HCC development.^[7-11] New technologies, such as global gene expression profiling, should allow to identify genes critical in early stages of tumorigenesis.^[12]

Oncogenes

Activated cellular oncogenes, particularly those of the ras family, have been found in a number of experimental hepatocarcinogenesis models. In human hepatocarcinogenesis, however, no consistent pattern of proto-oncogene activation has emerged so far for HCC. It is also of interest that no structural or functional changes of a large panel of oncogenes have been found in a transgenic mouse model which is believed to resemble the process of human hepatocarcinogenesis.

Tumor suppressor genes

Restriction fragment length polymorphism studies of paired HCC and non-tumorous liver samples have revealed relatively frequent (>20% in ¡Ý10 informative cases) chromosomal allelic losses (loss of heterozygosity, LOH) in HCC on chromosomes 4, 5q, 8p, 10q, 11p, 13q, 16, 16p, 16q, 17p, and 22q, suggesting that these sites may harbor tumor suppressor genes involved in the pathogenesis of HCC. In general, these genetic alterations appear to occur at later stages of HCC development. Interestingly, a G to T mutation at the third base position of codon 249 of the p53 gene, leading to a substitution of arginine to serine, was found in a significant number of HCC in patients from southern Africa and the Qidong area in China. It was suggested that this hot spot mutation was associated with high AFB1 intake in food and may have contributed to the high incidence of HCC in these areas. This finding was supported by in vitro studies indicating that the third base of codon 249 of p53 was preferentially targeted to form adducts with AFB1.

DNA mismatch repair genes

In addition to oncogenes and tumor suppressor genes, DNA mismatch repair genes have recently been identified as a new class of susceptibility genes involved in the pathogenesis of inherited and sporadic human tumors, most notably hereditary nonpolyposis colorectal cancer (HNPCC). Defective DNA mismatch repair can lead to the accumulation of mutations and microsatellite instability in the cellular genome and thus increase the chance of malignant transformation. The role of DNA mismatch repair defects in HCC development is currently unknown. Recent observations, however, suggest that the HBV x (HBx) gene/antigen may interfere with components of the DNA repair machinery. Telomerase activation The progressive shortening of chromosome ends, or telomeres, accompanies normal cell division and may contribute to cellular aging and serve as a control mechanism against unregulated cellular proliferation. Recently, a remarkable correlation between certain types of cancer and the expression of telomerase, a ribonucleoprotein enzyme preventing the shortening of telomeres, was found. Indeed, expression of telomerase may be a common pathway leading to cancer. In this regard, telome-rase activity was found in 85% of HCC tissues in a recent study.

Growth factors

As in most other forms of cancer, the unregulated expression of growth factors and of components of their signalling pathways may play an important role in hepatic oncogenesis. Indeed, overexpression of certain growth factors was found in HCC, including insulin-like growth factor II (IGF-II), transforming growth factor (TGF), hepatocyte growth factor (HGF), and insulin receptor substrate 1 (IRS-II). Surprisingly, HGF has been shown to inhibit the growth of a number of hepatoma cell lines and no neoplasms developed in transgenic mice expressing HGF in the liver under control of the albumin promoter. Moreover, in a recent study in c-myc-HGF double-transgenic mice it was found that coexpression of HGF markedly reduced c-myc-induced neoplastic changes. The knowledge regarding growth factors in HCC, however, is still incomplete and additional factors are likely to emerge as potentially important candidates involved in hepatocarcinogenesis.

Other factors

Other host factors beyond the ones addressed above may be genetic polymorphisms of enzymes metabolizing environmental xenobiotics, such as alcohol, benzpyrene and other polycyclic aromatic hydrocarbons in tobacco smoke, AFB1 and others.^[10,11,13] Using oligonucleotide or cDNA microarray expression profiling, suppression subtractive hybridization, proteomic analysis and other methods should allow to further elucidate the genetic events underlying HCC pathogenesis and to identify novel diagnostic markers as well as therapeutic targets.^[12,14-17]

Natural course of HCC

The prognosis of HCC patients is generally very poor. Most studies report a five-year survival rate of less than 5% in symptomatic HCC patients. Furthermore, these tumors have been shown to be quite resistant to radio- or chemotherapy. Investigations of the natural history and clinical course of HCC revealed a long-term survival of patients only with small asymptomatic HCC that could be treated surgically or nonsurgically.^[18,19] Most patients (>80%) are inoperable at the time of diagnosis. The prognosis of patients with inoperable HCC is very poor, their estrogen receptor status is the strongest prognostic factor for survival.^[4] Despite progress in early diagnosis and surgical or nonsurgical strategies, the overall survival of HCC patients has not significantly improved during the last two decades.^[5] Apart from developing novel therapeutic concepts, prevention strategies, therefore, are of paramount importance and need to be actively explored in order to reduce the incidence of HCC.

Gene therapy of HCC

Treatment strategies for HCC include surgical and nonsurgical interventions.^[19,20] Gene therapy for HCC is also being explored in vitro as well as in preclinical models.^[21,22] Gene therapy involves three concepts: gene substitution, gene augmentation and DNA vaccination.^[23,24] In addition, oncolytic viruses, anti-angiogenic strategies and others are being studied (Fig. 1).

Gene substitution

One of the most intriguing concepts of gene therapy for cancer is restoration of tumor suppressor functions, for example by introduction of the wild-type p53 tumor suppressor gene into tumor cells by various gene transfer strategies. Mutations of the p53 tumor suppressor gene are frequently found in HCC, especially in geographic regions where HBV infection and exposure to AFB1 are risk factors. Loss of wild-type p53 protein function is associated with the malignant phenotype via a specific growth or survival advantage for liver cells carrying the p53 mutation and enhances the cellular resistance to a variety of chemotherapeutic drugs.^[25,26] Conversely, introduction of a wild-type p53 gene into HCC cells carrying a mutated p53 gene may result in growth inhibition and restoration of sensitivity to chemotherapeutic drugs. This strategy has been successfully explored in vitro by retrovirus mediated transfer of wild-type p53 gene into human HCC cells, resulting in tissue-specific growth inhibition and chemosensitivity to cisplatin.^[27] A recent study showed that fusion of VP22 and p53 greatly improves the results of p53 gene substitution therapy.^[28]

Gene augmentation

Gene augmenation is aimed at the local expression of a therapeutic gene product that is physiologically not expressed or expressed at therapeutically insufficient levels. Apart from the expression of cytokines, e.g., interleukin-2 (IL-2) or tumor necrosis factor-alpha (TNF-¦Á), the therapeutic principle may be a ¡®suicide gene¡¯.

Complete regression of a murine HCC has been demonstrated in vivo by TNF-¦Á,^[29] by IL-2^[30] as well as by an activatable interferon regulatory factor-1 in mice.^[31] Gene transfer can be achieved in vivo by delivering retroviral^[29] or adenoviral vectors^[30] systemically, directly into the tumor or into the peritoneal cavity. In principle, gene transfer can also be performed ex vivo^[32] by transducing tumor infiltrating lymphocytes (TILs) from the patient¡¯s HCC with the therapeutic gene, expanding the TILs in vitro and giving the ex vivo modified TILs back to the patient.

Another strategy to treat HCC is genetic prodrug activation therapy via the introduction of a ¡®suicide gene¡¯ into malignant cells followed by the administration of the prodrug. This concept has been experimentally explored in HCC cells in vitro and in vivo, e.g., for the HSV-tk gene,^[33-37] the gene encoding cytosine deaminase (CD) that converts the prodrug 5-fluorocytosine to 5-fluorouracil that inhibits RNA and DNA synthesis during the S-phase of the cell cycle,^[38] the gene encoding purine nucleoside phosphorylase that converts purine analogs into freely diffusible toxic metabolites,^[39,40] and the gene encoding cytochrome p450 4B1.^[41]A significant bystander effect of cell killing caused by suicide gene expression could be demonstrated in vitro and in vivo, based on cell-cell contact rather than release of cytotoxic substances from the transduced cells.^[42] At the same time, the bystander effect may also affect non-malignant dividing cells in the target tissue, potentially limiting the application of this strategy.

DNA vaccination

An elegant and innovative application of gene therapy is the manipulation of the immune system by introduction of expression vectors into muscle cells, resulting in long lasting cellular and humoral immune responses.^[43-46] DNA-based tumor vaccination against HCC may be possible, for example, by intramuscular introduction of a plasmid expressing HCC-specific antigens or antigens that are highly overexpressed in HCC cells, such as AF-20 antigen, insulin receptor substrate-1,^[47] alpha-fetoprotein,^[48] aspartyl asparaginyl hydroxylase, mutated p53 protein and others. Potential limitations of this strategy include the regulation of the immune response as well as the low level expression of the targeted antigen in non-malignant cells,^[49] rendering them susceptible to immune mediated elimination as well.

Oncolytic viruses

A new and elegant approach is the use of viruses that lyse tumor cells. In view of the frequent p53 mutations in HCC, p53 mutations were used for the selective, adenovirus-mediated lysis of tumor cells. Thus, an adenovirus mutant was engineered to replicate selectively in p53-deficient^[50-52] or AFP-expressing human tumor cell lines.^[53] Apart from adenoviruses, genetically engineered herpesviruses are also used.^[54]

Other strategies

Anti-angiogenic gene therapy aimed at inhibiting the formation of new blood vessels is another attractive concept based on the expression of angiostatin,^[55,56] endostatin,^[57,58] antisense constructs against vascular endothelial growth factor (VEGF),^[59] soluble VEGF receptor^[58,60] or a mutant Raf gene that blocks endothelial signaling and angiogenesis in response to multiple growth factors.^[61]

HCC prevention

HCC prevention includes primary prevention aimed at preventing chronic liver diseases of different etiological causes and secondary prevention aimed at preventing the recurrence and/or the development of new HCC lesions after successful surgical or nonsurgical treatment of HCC.

Primary HCC prevention

Apart from developing and refining novel therapeutic strategies, the implementation of measures for the primary prevention of HCC development is most important. Primary prevention is aimed at the interference with HCC development at four stages (Fig. 2).

Stage 1

Interventions at this step are aimed at the prevention of acquired liver diseases. Apart from avoiding liver toxins including alcohol and certain drugs or infections with HBV or HCV by hygienic measures, it is also important to avoid parenteral exposure to blood, blood products or contaminated needles. A prime example for HCC prevention is vaccination against HBV infection using the commercially available active and passive vaccines. Several HBV vaccines using natural or recombinant hepatitis B surface antigen (HBsAg) from different sources are well introduced in clinical practice, and universal vaccination in Taiwan has indeed already resulted in a decline of the incidence of HCC.^[62] In addition, novel HBV vaccination strategies are being explored, including a novel triple HBsAg recombinant vaccine,^[63] epidermal HBsAg powder immunization^[64] as well as oral immunization using HBsAg transgenic plants.^[65-67] Further, DNA vaccination has been shown in animal models to induce antibodies against HBsAg (anti-HBs),^[68,69] even after topical application to the skin. For the prevention of HCV infection, however, there is no effective vaccine available to date. While several HCV vaccination concepts are being evaluated, including HCV proteins,^[70] HCV-like particles^[71] as well as intravenous, intrahepatic, intra-epidermal, intramuscular or oral cDNA immunization,^[72-76] it is not to be expected that a vaccine protecting against HCV infection will become commercially available within the next few years.

Stage 2

Interventions at this step are aimed at the early treatment of acute liver diseases, thereby blocking their transition into chronic hepatitis that carries the risk for developing liver cirrhosis and its sequelae, including HCC development. While the principles mentioned above regarding liver toxins are also applied here, the early diagnosis and treatment of inherited liver diseases, such as Wilson¡®s disease and hemochromatosis, are of paramount importance. Further, a recent study suggests that early treatment of acute HCV infection prevents its progression to chronic hepatitis C in more than 90% of patients.^[77]

Stage 3

Interventions at this step are aimed at the prevention of the progression of chronic hepatitis to liver cirrhosis that carries a high risk for HCC development. Apart from avoiding liver toxins, the treatment of chronic hepatitis is here most important. This includes the treatment of inherited, cholestatic or autoimmune liver diseases as well as the treatment of chronic viral hepatitis B or C. Reduction of iron overload by phlebotomy, for example, has been shown to stop the progression of hemochromatosis to liver cirrhosis and HCC. Treatment of chronic hepatitis B by interferon alpha or nucleoside analogs^[78-80] and chronic hepatitis C by interferon alpha and more recently by its combination with the nucleoside analogue ribavirin demonstrated biochemical, virological and histopathologial improvement^[81-83] and a lower incidence of HCC development.^[84-86] Based on the concept of immune pathogenesis of HCC,^[6] it has recently been shown that treatment of animals with anti-fas ligand antibodies prevents HCC development associated with chronic hepatitis B.^[87,88]

Stage 4

Interventions at this step are aimed at interfering with the molecular events leading to HCC development, usually in a cirrhotic liver. These strategies include all treatment modalities detailed above (stage 3) as far as they can be implemented in patients with compensated or decompensated liver cirrhosis. In addition, some of the measures to pevent HCC recurrence after successful HCC treatment (secondary prevention, see below) should in principle also be useful for HCC prevention at this stage of the liver disease. Further, some concepts of molecular therapy of HCC (see above) should be applicable also for the prevention of HCC. Without experimental preclinical data on these issues it would be premature, however, to discuss their potential clinical impact.

Secondary HCC prevention

The prevention of a local recurrence and/or the development of new HCC lesions in patients after successful surgical or nonsurgical treatment of HCC (Fig. 3) is of paramount importance in improving disease-free and overall survival rates of patients.

Stage 1

HCC treatment strategies include surgical as well as nonsurgical interventions. Surgical interventions are resection and in selected cases liver transplantation.^[89-92] Because the majority of patients present with advanced HCC at the time of diagnosis or carry a high surgical risk due to comorbidities or advanced age, nonsurgical interventions are of great clinical importance. These include percutaneous ethanol injection (PEI),^[93,94] percutaneous acetic acid injection (PAI),^[95] percutaneous thermal ablations, e.g., radiofrequencey thermal ablation (RFTA),^[96-99] laser induced thermal ablation (LiTT)^[100] as well as transarterial chemotherapy (TAC), transarterial chemoembolisation (TACE)^[93,101-103] or transarterial 131-iodine lipiodol therapy. In addition, a number of drugs have been evaluated in clinical trials.^[104,105] Several agents that have been shown not to be effective in randomized controlled clinical trials have yielded equivocal or inconsistent results or have not been evaluated in large qualified clinical trials. They include tamoxifen^[106-108] octreotide,^[109-111] thymostimulin,[^112] pravastatin^[113] and gemcitabine.^[114-116] These substances deserve further clinical evaluation. Overall, however, the only potentially curative treatment is surgery, especially liver transplantation. HCC resection and some nonsurgical interventions, especially PEI and RFTA, can in selected cases prolong disease-free as well as overall survival with a survival rate of about 50% at 5 years and in a few cases even result in a long-term remission or cure.

 Stage 2

After successful HCC resection or nonsurgical ablation, HCC recurrence in the remaining, usually cirrhotic liver, is the major limitation of the life expectancy of these patients. The probability of recurrence is about 50% within 3 years after successful treatment.^[91,117] Strategies to prevent HCC recurrence are, therefore, focused on the improvement of survival of HCC patients after initial cure. The strategies explored to date include the administration of polyprenoic acid, an acyclic retinoid,^[118] of interferon alpha^[119] and of interferon beta.^[120] Further, adoptive immunotherapy^[121] and intraarterial 131-iodine lipiodol treatment^[122] have been evaluated in clinical studies. All these interventions have resulted in a lower recurrence rate of HCC. These findings have to be confirmed, however, in larger randomized controlled studies demonstrating a clear clinical benefit for secondary prevention.

Summary and perspectives

HCC is one of the most common malignant tumors in some areas of the world with an extremely poor prognosis. The major etiologic risk factors for HCC development include toxins (alcohol, AFB1), HBV and HCV infection as well as various inherited metabolic disorders, such as hemochromatosis.

The molecular pathogenesis of HCC development is very complex and involves alterations in the structure or expression of several tumor suppressor genes, oncogenes and, possibly, mechanisms leading to a genetic instability due to mismatch repair deficiency or chromosomal instability and aneuploidy due to defective chromosomal segregation. Central to the molecular pathogenesis of HCC are mutations of various genes and a genetic instability which in most cases result from chronic liver disease and the associated enhanced liver cell regeneration and mitotic activity.

Gene therapy for HCC falls into three categories: gene replacement, gene augmentation and DNA vaccination. In view of the complexity of the genetic events underlying hepatocarcinogenesis, most studies performed to date have focused on gene augmentation as an experimental therapeutic strategy. Despite exciting prospects of nucleic acid-based therapy of HCC, various aspects of delivery, targeting and safety need to be addressed before these concepts will enter clinical practice.

Apart from improving HCC therapy, the refinement and implementation of existing as well as the development of novel strategies aimed at HCC prevention are most important. Primary prevention has been shown to reduce HCC development in some risk groups. For example, hepatitis B vaccination of children in Taiwan has actually resulted in a decline of the HCC incidence. Further, antiviral therapy of patients with chronic hepatitis B or C should contribute to HCC prevention. Public health measures to reduce food contamination with AFB1 and eliminate excessive alcohol use should also reduce the incidence of chronic liver disease, cirrhosis and thereby HCC. Apart from primary HCC prevention, interventions aimed at secondary prevention after successful HCC treatment are also a very active area of basic and clinical research. Preventive measures should have a major impact on reducing the incidence and the recurrence of HCC, one of the most common and devastating malignancies in the world.

Competing interest

 No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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Received December 12, 2002

Accepted after revision December 19, 2002