The prognostic molecular markers in hepatocellular carcinoma

Abstract

The prognosis of hepatocellular carcinoma (HCC) still remains dismal, although many advances in its clinical study have been made. It is important for tumor control to identify the factors that predispose patients to death. With new discoveries in cancer biology, the pathological and biological prognostic factors of HCC have been studied quite extensively. Analyzing molecular markers (biomarkers) with prognostic significance is a complementary method. A large number of molecular factors have been shown to associate with the invasiveness of HCC, and have potential prognostic significance. One important aspect is the analysis of molecular markers for the cellular malignancy phenotype. These include alterations in DNA ploidy, cellular proliferation markers (PCNA, Ki-67, Mcm2, MIB1, MIA, and CSE1L/CAS protein), nuclear morphology, the p53 gene and its related molecule MDM2, other cell cycle regulators (cyclin A, cyclin D, cyclin E, cdc2, p27, p73), oncogenes and their receptors (such as ras, c-myc, c-fms, HGF, c-met, and erb-B receptor family members), apoptosis related factors (Fas and FasL), as well as telomerase activity. Another important aspect is the analysis of molecular markers involved in the process of cancer invasion and metastasis. Adhesion molecules (E-cadherin, catenins, serum intercellular adhesion molecule-1, CD44 variants), proteinases involved in the degradation of extracellular matrix (MMP-2, MMP-9, uPA, uPAR, PAI), as well as other molecules have been regarded as biomarkers for the malignant phenotype of HCC, and are related to prognosis and therapeutic outcomes. Tumor angiogenesis is critical to both the growth and metastasis of cancers including HCC, and has drawn much attention in recent years. Many angiogenesis-related markers, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PD-ECGF), thrombospondin (TSP), angiogenin, pleiotrophin, and endostatin (ES) levels, as well as inratumor microvessel density (MVD) have been evaluated and found to be of prognostic significance. Body fluid (particularly blood and urinary) testing for biomarkers is easily accessible and useful in clinical patients. The prognostic significance of circulating DNA in plasma or serum, and its genetic alterations in HCC are other important trends. More attention should be paid to these two areas in future. As the progress of the human genome project advances, so does a clearer understanding of tumor biology, and mo re and more new prognostic markers with high sensitivity and specificity will be found and used in clinical assays. However, the combination of some items, i.e., the pathological features and some biomarkers mentioned above, seems to be more practical for now.

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INTRODUCTION

Liver cancer is one of the common malignancies worldwide, and has been ranked the 2^nd cancer killer in China since the 1990s. Although many advances in the clinical study of hepatocellular carcinoma (HCC) have been made, and long-term survival of patients has been obtained in some clinical centers, only a definitive subset of cases is cured by surgery, and the overall dismal outcome of patients with HCC has not been completely changed. Lack of control of metastatic foci and recurrence are the most prevalent causes of death in patients with HCC, and it is important for tumor control to identify the factors that predispose patients to death. Much effort has been made to predict HCC behavior, but specific prognostic indicators are still lacking[1].

Prognostic factors in HCC conventionally consist of staging with the tumor node metastasis system (TNM) and grading by tumor cellular differentiation. There are also other factors useful in prognostic predication but most of them are clinic al. With new discoveries in cancer biology, pathological and biological factors of HCC in relation to prognosis have been studied quite extensively. Morphologic al features of the tumor, both gross and histological, have been found to significantly associate with tumor recurrence and patient survival[2-4]. A complementary way is to analyze molecular markers for their prognostic significance with reference to tumor recurrence and survival term in HCC. A large number of molecular biological factors have been shown to associate with the invasiveness of HCC, and have potential prognostic significance. However, routine biomarkers for the prediction of HCC prognosis are not yet available. In this review, we will focus on the recent advances in this aspect.

Cellular malignancy is a very important aspect for patient prognosis. In recent years, with the development of cellular and molecular biological techniques, many molecular markers related to invasion, metastasis, recurrence and survival have been explored. In HCC, DNA ploidy, the proliferating activity of tumor cells, tumor suppressor and promoter genes, cell cycle controllers, proteinases that degrade extracellular matrix, adhesion molecules, angiogenic factors, and metabolic genes, have been regarded as biomarkers for the malignant phenotype of HCC, and are related to prognosis and therapeutic outcomes[5].

CELLUAR MALIGNANCY-RELATED MARKERS

DNA-ploidy

Controversy still exists regarding the prognostic significance of DNA ploidy in HCC patients. Many reports indicate that DNA ploidy could be a predictive marker for HCC prognosis[6,7]. The overall survival rate of patients with aneuploid cells is much lower than that of patients with diploid ones, and those wit h multiple G0/G1 peaks have the worst prognosis. Patients with higher cell proportions in proliferating stages have a higher early recurrence rate[7]. However, other studies could not find a relationship between DNA ploidy and prognosis.

Proliferating activity of HCC cells

Many antigens, such as proliferating cell nuclear antigen (PCNA), Ki-67, Mcm2, MIB1, MIA, and CSE1L/CAS protein (CAS), have been used as proliferation markers for cancer cells. The detection of PCNA with immunohistochemical methods is a common way to study the proliferating activity of cancer cells[8]. Combine d with histopathological characteristics, the PCNA labeling index (PCNA-LI) is one useful marker for evaluating malignant grade, and for predicting recurrence time and the patients’ prognosis of HCC[9].

The expression of the human Ki-67 protein is strictly associated with cell proliferation. The fact that the Ki-67 protein is present during all active phases of the cell cycle, but is absent from resting cells, makes it an excellent marker for determining the so-called growth fraction of a given cell population. Ki-67 protein expression is an absolute requirement for progression through the cell-division cycle. The fraction of Ki-67-positive tumor cells (the Ki-67 labeling index) is often correlated with the clinical course of the disease[10,11]. Higher Ki-67 labeling index (Ki-67-LI) has a very similar clinical significance to PCNA-LI, reflecting the existence of biologically aggressive phenotypes and poor overall and disease-free survival rates in HCC. This could be a useful factor for predicting the long-term survival of patients with HCC following hepatic resection[12,13].

The CSE1L/CAS protein (CAS) is a Ran-binding protein with a function as a nuclear transport (export) factor. This protein plays a role in the mitotic spindle checkpoint, which assures genomic stability during cell division. This checkpoint is frequently disturbed in neoplasias of various origins, including hepatic tumors. The degree of CAS expression correlates with the grade of tumor dedifferentiation, and could be a prognostic marker for HCC[14].

Nuclear morphology

Nuclear profiles have been reported as useful prognostic predictors in various cancers, including HCC. The nuclear area of HCC correlates with cell differentiation and cell proliferating activity, and HCC with a large nuclear area has high potential for blood vessel invasion and intrahepatic metastasis. Computerized nuclear morphometry is more objective and quicker than conventional microscopic analysis[15]. Recently, quantitative nuclear morphometry of cancer cells followed by computer-assisted image analysis (termed Quantitative nuclear grade, QNG) has proven to have potential use in cancer detection and predicting outcomes such as tumor stage, recurrence, and progression[16].

p53 gene and its related molecule MDM2

P53 protein plays a central role in cellular responses, including cell-cycle arrest and cell death in response to DNA damage. p53 dysfunction can induce abnormal cell growth, increased cell survival, genetic instability, and drug resistance. Mutations in the p53 gene are the most frequently reported somatic gene alteration in human cancer. Associations of p53 mutation or positive immunohistochemistry staining with higher grade and more advanced stage has been noted for cancers of various origins. In addition, p53 mutation is considered as a strong marker predicting an increased risk of local relapse, treatment failure, and overall and disease-free survival in many kinds of human carcinomas, such as breast[17-19], colorectal[20], esophageal[21], head and neck[22], lung[23,24], and ovarian[25], as well as sarcoma[26]. An increased intracellular concentration of the P53 protein, although no t identical to, is sometimes seen in tumors with p53 mutation, and has been correlated with poor prognosis in some tumor types. Several studies have shown a relationship between the nuclear accumulation of p53 protein and poor disease-free and overall survival of cancer patients[27,28]. The presence of serum a nti-p53 antibody has also been shown to associate with survival of patients wit h breast, ovarian, and colorectal cancer[29,30]. p53 mutations in plasma DNA could also be detected in cancer patients, and may be used as a prognostic factor and an early marker to indicate recurrence or distant metastasis[31]. However, there is still a great controversy as to whether alteration of the p53 gene adversely affects survival of cancer patients. Many reports failed to show the independent prognostic value of p53 in the carcinomas of tongue[32], breast[33,34], stomach[35], lung[36], ovarian[37], bladder[38], colorectal[39], and non-Hodgkin’s lymphoma[40].

In a similar situation, there are many very controversial results with the prognostic value of p53 overexpression or p53 gene mutation in HCC patients. Many stu dies showed that p53 mutation was involved in determining the dedifferentiation, the proliferating activity, and tumor progression[41], was strongly rel ated to the invasiveness of HCC, and may also influence the postoperative course (particularly the recurrence within 1 year)[42,43]. Mutations in the p53 gene or positive immunostaining for mutant P53 protein expression could be use d as a significant indicator of poor prognosis. HCCs with p53 mutations have a h igh malignant potential, and p53 mutation in the primary lesion is useful as an indicator for the biological behavior of recurrent HCCs. It is also a useful independent prognostic factor affecting survival after recurrence[9,44,45].

In a recent prospective study, we found the 3-year and 5-year overall survival rates of HCC patients with positive P53 nuclear accumulation were much lower than those of the HCC patients with negative P53 expression. In univariate and multivariate Cox analysis, p53 overexpression was the most significant factor that associated with the overall survival rates of HCC patients after resection. Its significance was even greater than that of factors such as tumor size, vascular invasion, and tumor capsule, though they were also related to the overall survival. p53 mutation or nuclear accumulation of p53 expression could be a valuable marker for predicting the prognosis of HCC patients after resection[46].

Serum anti-p53 antibody also could be a useful prognostic factor for HCC patients[47]. However, many different results showed that neither the immunohi stochemical detection of p53 expression, nor the serum anti-p53 antibodies had a significant prognostic value for outcome of patients with HCC[48,49].

The transcription of the mdm2 gene is activated by p53 and this limits the growth-suppressing activity of p53 by direct binding. It has been reported that MDM2 protein is overexpressed in several types of cancers. Endo found MDM2 overexpression correlated positively with p53 mutation, and is a useful predictor of poor prognosis in patients with HCC following hepatic resection[50].

Cell cycle regulators

Disruption of the G1/S and G2/M check points leads to uncontrolled cell growth, resulting in the development and progression of cancers. Overexpression of cyclin A, cyclin D, and cyclin E have been found to correlate with the tumor relapse of human HCC, and are independent predictive markers for their recurrence and prognosis[51,52]. The enhanced expression of cyclin E correlates with hype rphosphorylation of pRb and a high frequency of Ki-67-positive cells. HCCs wit h enhanced cyclin E expression probably contain a relatively large number of pro liferating cancer cells[52]. cdc2 overexpression seems to play the most crucial role of the modulators in cell cycle progression and cell proliferation of HCC, and significantly predicts recurrence[53].

The p27 protein binds and inhibits cyclin/cyclin-dependent kinase complexes, is a negative regulator of cell-cycle progression. The central role of p27 makes it important in a variety of disease processes, particularly in neoplasia, that involve aberrations in cellular proliferation and other cell fates. Loss of p27 cooperates with mutations in several oncogenes and tumor suppressor genes to facilitate tumor growth, indicating that p27 may be a "nodal point" for tumor suppression. In most tumor types, reduced p27 expression correlates with poor prognosis, making p27 a novel and powerful prognostic marker[54]. High p27 expression, correlated with prolonged survival, is a favorable independent progno stic parameter for HCC[55,56].

The protein p73, the first identified homologue of p53 gene, has been shown to i nduce apoptosis. P73 expression status is significantly related to prognosis of HCC patients, and could serve as a useful indicator of prognosis in HCC patients[57]. There is still controversy with the prognostic value of the p16IN K4a and p15INK4B genes[58].

Tumor promoter genes and their receptors

Aberrations of many tumor promoter genes, such as ras, c-myc, c-fms have been indicated as indicators of malignant potential and poor prognosis in HCC[9,59-61]. c-myc amplification and p53 alteration may be copa rticipating events in the progression of HCC. Disease-free survival in patients showing c-myc amplification is significantly shorter than in those without amplification. Hepatocyte growth factor/scatter factor (HGF/SF) is one of the most important hu moral mediators of liver regeneration. It is potentially related to molecular mechanisms of hepatocarcinogenesis via a paracrine system involving its cellular receptor, c-met. Up-regulation of c-met plays an important role in the development and progression of HCC, and may be a prognostic marker. Its express ion level is inversely correlated with survival coordinated with uPA expression[62,63]. However, there is no significant correlation between the HGF le vel in tumor and the survival rate of HCC patients[5].

Among of the erb-B receptor family members, c-erbB-2 (Her-2/neu) represents a well-established prognostic marker and therapeutic target in several human tumor types, especially breast cancer. However, c-erbB-2 is neither a prognostic marker nor a relevant therapeutic target in human HCCs[9,64]. EGF-R and c-erbB-3 play important roles in the progression of HCC, affecting disease-free survival of HCC patients[5,65].

ets-1 has also been shown to link to cancer invasion and metastasis. ets-1 expression was observed with high incidence. However, the average labeling index (LI) in HCC is lower than in noncancerous lesions. Even lower expression levels we re found in HCCs of high TNM stage, poor differentiation, portal invasion, intra hepatic metastasis, large tumor size, and high Ki-67-LI. HCC patients with highets-1 expression showed better outcomes for disease-free survival than those with low ets-1 expression[66].

Apoptosis related

The expression of Fas and Fas ligand (Fas L) play a role in apoptosis of cancer cells including HCCs, and associates with the prognosis of cancer patients. Fas expression level is significantly decreased in poorly differentiated HCC and of large size, while Fas L expression in carcinoma cells is observed exclusively in moderately or poorly differentiated cases. Each of them has prognostic significance for disease-free survival (DFS)[5,67,68].

Telomerase activity

The ribonucleoprotein telomerase extends telomeres in cancer cells and has been proposed as a prognostic marker for cancer. Telomerase activity can be identified as an independent predictor for recurrence after resection of HCC[69]. The peripheral blood telomerase activity can also be used as a molecular marker for the detection of circulating hepatoma cells in blood of HCC patients, which reflect haematogenous micrometastasis. This is potentially a practical diagnostic/predictive marker of HCC[70]. Quantitative analysis of telomerase activity shows that the patients with positive telomerase activity in noncancerous liver tissue have a higher recurrence rate after HCC resection. The relative telomerase activity (RTA) of early recurrent patients is significantly higher than those without recurrence. So, RTA could be a predictive marker for early recurre nce after HCC resection[71].

CELL ADHESION AND EXTRACELLULAR MATRIX RELATED

Adhesion molecules

The expression level of E-cadherin inversely correlates with HCC histological grade and prognosis. E-cadherin underexpression might have some contribution to the early recurrence of HCC[72,73]. In contrast, alpha-, beta-, and ga mma-catenin expression significantly correlated positively with HCC grade, bein g the highest in poorly differentiated HCC. Significant positive associations were found between gamma-catenin high expression and capsular invasion or presence of satellite nodules, and between beta-catenin high expression and vascular invasion. HCC patients with underexpression of E-cadherin, alpha-catenin, and gamma-catenin, and patients with overexpression of beta-catenin, had poorer survival rates[73]. HCCs with a nonnuclear type of beta-catenin overexpres sion were frequently larger than 5 cm in diameter and had poorer cellular diff erentiation, more invasiveness, and the patients had significantly shorter disea se-free survival lengths[74,75]. beta-catenin mutation associates with nuclear expression of the protein, and is a favorable prognostic factor related to low stage[76].

Serum concentration of intercellular adhesion molecule-1 (sICAM-1) in patients with HCC is a marker for disease progression and prognosis. Higher sICAM-1 levels are more frequently observed in those patients with multiple lesions and int rahepatic metastasis, and their prognosis is also very poor. Detecting sICAM-1 is of important value in predicting tumor recurrence after surgery[77-79]. The CD44 proteins form a ubiquitously expressed family of cell surface adhesion molecules involved in cell-cell and cell-matrix interactions. The major physiological role of CD44 is to maintain organ and tissue structure via cell-cell an d cell-matrix adhesion, but certain variant isoforms can also mediate lymphocyte activation and homing, and the presentation of chemical factors and hormones. The expression of multiple CD44 isoforms is greatly upregulated in neoplasia. CD 44, particularly its variants, may be useful as a diagnostic or prognostic marker of malignancy in at least some human cancers[80]. Up-regulation of CD 44 isoforms such as CD44s, CD44v5, CD44v6, CD44v7-8, and CD44v10, correlates with high histological grade, being the highest in poorly differentiated HCC. CD44 s positivity was an independent factor. Positivity for one or more CD44 isoforms was the most useful independent factor for overall survival[81].

Degradation of extracellular matrix

The matrix metalloproteinases (MMP) and the plasminogen activation system (PA) p lay crucial roles in the process of cancer invasion and metastasis. Their expression levels were found correlated to recurrence and survival after HCC resection[82,83].

MMP-2, MMP-9, and tissue inhibitors of metalloproteinases-1, -2 (TIMP-1, T IMP-2) have been found to be of prognostic significance in HCC. The content of MMP-2, MMP-9 in HCC being higher than that in surrounding liver parenchyma could be used as an important index to judge the invasion and metastasis of HCC[84,85]. Plasma MMP-9 levels can also be a candidate for a novel marker for HCC. The levels appear to reflect the potential and ongoing activity of vascula r invasion[5].

In several tumor types, elevated levels of urokinase plasminogen activator (uPA), its receptor (uPAR) or its inhibitor plasminogen activator inhibitor-1 (PAI-1) is associated with a poorer prognosis[85]. uPA activity may be the mo st sensitive factor affecting HCC invasion in the plasminogen activation system and is a strong predictor for the recurrence and prognosis of HCC[86,87]. The PAI-1 proteinis a multifaceted proteolytic factor. It not only functions as an inhibitor of the protease uPA, but also plays an important role in signal transduction, cell adherence, and cell migration. Thus, an apparent paradox considering its name-although it inhibits uPA during blood coagulation, it actually promotes invasion and metastasis. In many malignancies including HCC, elevated PAI-1 is associated with tumor aggressiveness and poor patient outcome[86].

ANGIOGENESIS RELATED

Tumor angiogenesis is critical to both the growth and metastasis of cancer, and is regulated by angiogenic factors. Circulating angiogenesis regulators have bee n evaluated not only as diagnostic and/or prognostic factors but also as predict ive factors in cancer patients. They could be used to determine the risk of developing cancer, to screen for early detection, to distinguish benign from malignant disease, and to distinguish between different types of malignancies. In established malignances, they can be uses to determine prognosis, to predict the response to therapy, and to monitor the clinical course[5,83,88]. HCC is typically a hypervascular tumor with a rich blood supply. In recent years, many angiogenesis-related markers, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-deriwed endothelial cell growth factor (PD-ECGF), thrombospondin (TSP), angiogenin, pleiotrophin and endostatin (ES) levels, as well as inratumor microvessel density (MVD) have been evaluated and found to relate to HCC prognosis.

Intratumor microvessel density (MVD)

The intratumor MVD is a direct reflection of tumor angiogenesis. It can be visua lized by immunohistochemical staining with antibodies to anti-CD34, Factor VIII, and alpha smooth muscle actin[89]. MVD levels have a close relationshi p with the tumor capsule status, tumor size (HCC with 2-5 cm in diameter has t he highest MVD level), intrahepatic recurrence and disease-free survival, and can be a predictive marker for disease-free survival[90]. In the authors institute, three types of intratumor microvessels, including capillary-like, sinusoid-like, and mixed-type, were found in HCC. The MVD level was not related to tumor size, capsule status, Edmondson’s grade, or alpha-fetoprotein level; was an independent factor of disease-free survival in small HCC patients; and was a predictive marker for early recurrence[91].

Vascular endothelial growth factor (VEGF)

A substantial number of studies have demonstrated a strong association between elevated tumor expression of VEGF and advanced disease or poor prognosis in various cancers. Circulating VEGF seems to be a reliable surrogate marker of angiogenic activity and tumor progression in cancer patients. It may be predictive of tumor status and prognosis in patients with different types of cancer, and may be useful in predicting and monitoring tumor response to anticancer therapies and in follow-up! surveillance for tumor relapse. It may provide new prognostic information that is not afforded by conventional clinicopathologic prognostic indicators[92]. In HCC, a high serum VEGF level significantly correlates with absence of tumor capsule, presence of intrahepatic metastasis, presence of microscopic venous invasion, and advanced stage, and it may be useful as a biologic marker of tumor invasiveness and a prognostic factor in HCC[93]. The data of the authors’ institute also shows serum VEGF is a predictor of invasion and metastasis of HCC and a potential biomarker of metastatic recurrence after curative resection[94,95].

Platelet-derived endothelial cell growth factor (PD-ECGF)

PD-ECGF may not be a major regulator of angiogenesis of HCC, but may play an important role in hepatocarcinogenesis, cooperating with hepatitis C virus. PD-EC GF expression associates with the venous invasion of HCC[96].

GENOMICS AND PROTEOMICS RELATED

Molecular genetic analyses have clarified that accumulation of genomic changes provides important steps in carcinogenesis and have identified a number of valuable genetic markers for certain cancers. The association of these genomic aberrations with the progression and prognosis of cancer has drawn more and more attention. To date, allelic loss of 1p, 1q21-23, 2p21-16.3, 3p24-p25, 8p22, 8p23, 9 p21, 9q, 10, 13q12, 17p13.3, and 22q13 have been proposed to be related to the survival and prognosis of cancer patients[97-101].

Many chromosomal aberrations, including gain of 1q, 8q, and 20q, and loss of 16q, 4q, 17p, 1p, and 8p have been identified in HCC[102]. However, the relationship between these recurrent alterations and the clinical phenotypes and prognosis is still unknown. Towards the end of 1999, we compared the differences of chromosomal aberrations between the primary HCC tumors and their matched metastatic lesions using a comparative genomic hybridization (CGH) technique, and found chromosome 8p deletions might contribute to HCC metastasis[103]. This result was further confirmed by comparison between nude mice models of HCC with different metastatic potentials[104]. In addition, a more accurate location was identified on 8p23.3, 8p11.2[105]. These findings provide new targets for exploring new predictive markers for the recurrence and prognosis of HCC. Recently, Itano et al[103] used restriction landmark genomic scanning (RLGS), a new high-speed screening method for multiple genomic changes, to detect un known genetic alterations in HCC. They found the disease-free survival r ate for patients with > or = 16 changed RLGS spots was significantly lower than that for patients with fewer changed RLGS spots (< or = 15 spots). In multivariate analysis, the number of changed spots was proven to retain an independent prognostic value. These results suggest that the number of changed RLGS spots may be a useful biological marker for recurrence of HCC[106].

One important trend in this area that should be paid attention to is the prognostic value of circulating DNA in plasma or serum, and its genetic alterations in cancer patients. Small amounts of DNA circulate in both healthy and diseased hum an plasma/serum, and increased concentrations of DNA are present in the plasma of cancer patients. Characteristics of tumor DNA have been found in genetic material extracted from the plasma of cancer patients. These features include decreas ed strand stability, the presence of specific oncogene or tumor suppressor gene mutations, microsatellite alterations, Ig rearrangements and hypermethylation of several genes. The results obtained in many different cancers have opened a new research area indicating that plasma DNA might eventually be a suitable target for the development of noninvasive diagnostic, prognostic and follow-up tests for cancer[107]. Blood testing for circulating tumor genetic markers may provide valuable prognostic information and guide future therapy[108].

However, there is still controversy over the prognostic significance[109]. We found loss of heterozygosity (LOH) on chromosome 14q (D14S62 and D14S51) could be detected in plasma DNA, and could be of prognostic significance in HCC patients[1].

Proteomics, regarded as a sister technology to genomics, is one of the technologies rapidly changing our approach to understanding tumor biology. By com paring the proteins present in diseased samples with those present in normal samples, it is possible to identify changes in expression of proteins that potentia lly may be related to tumor progression, invasion and metastasis, and prognosis. This technique has now made it possible to analyze proteins using high throughput, automated techniques. Proteomic profiling can be applied to tissue samples as well as body fluids (e.g. serum, urine, etc.), and it can provide surrogate markers of disease processes, potential response to treatment, possibility of recurrence and metastasis for cancers including HCC[110].

OTHERS

In addition, higher levels of urinary TGF-beta 1[111], heat shock protein-27 (HSP-27)[112] and Glutamine synthetase (GS) expression in the tumor[113], increased levels of cyclooxygenase-2 (COX-2) in nontumor liver tissue[114], preoperative serum IL-10[115], and HFE mutation s[116] or down-regulation of DRH1[117] are also powerful prognostic indicators for shorter disease-free survival and poor prognosis, related t o tumor progression of HCC.

The RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) gene suppresses the invasive and metastatic activities of cancers, has negative effects on the invasiveness of HCC, and can be regarded as a promising prognostic molecular marker for HCC[118].

EXPERIENCES OF THE AUTHORS’ INSTITUTE

At the authors’ institute, many molecular factors have been investigated and found to be related to HCC invasiveness in recent years. They could be divided into two groups: one is positive invasiveness-related factors, including p16 and p53 mutations, H-ras, c-erbB2, mdm2, TGF-a, epidermal growth factor receptor (E GFR), MMP-2, uPA, uPA-R and PAI-1, ICAM-1, VEGF, PD-ECGF, bFGF, and osteopo ntin (OPN), etc. The other group is negative invasiveness-related factors, including nm23-H1, Kai-1, TIMP-2, integrin a5, E-cadherin, etc. These factors could be potential predictive markers for the prognosis of HCC. Serum ICAM-1 and PAI-1 levels were higher in patients with metastasis than those without metastasis, while serum Thrombomodulin concentration negatively associated with the in trahepatic spreading and portal vein thrombi of HCC. Deletions of chromosome 8p and 17p, overexpression of MMP-2, TGF-a, and EGFR in HCC tissues, and LOH on chromosome 14q (D14S62 and D14S51) in plasma DNA were also related to metastatic recurrence and prognosis of HCC patients. p53 mutation or nuclear accumulation of p53 expression could be a valuable marker for predicting the prognosis of HCC patients after resection. E-cadherin, nm23, TIMP-2 are promising prognostic ma rkers[1,43,46,77,84,86,91,94-96,104-106].

To search for metastasis-associated genes on a global genomic scale, we recently used cDNA microarrays containing approximately 9984 human transcripts to investigate the gene expression profiles of primary tumors and their corresponding metastatic lesions (intrahepatic metastasis or tumor thrombosis of portal vein). A total of 79 significantly upregulated and 69 downregulated genes were identified. Some of them have proven to promote HCC metastasis[119]. These will provide new prognostic markers for predicting the possibillity of metastatic recurrence and survival after operation.

QUESTIONS AND PROSPECTS

In summary, pathologic factors indicative of tumor invasiveness such as tumor size, number, capsule state, venous invasion, presence of satellite nodules, and advanced pTNM stage, are the best-established risk factors for recurrence and important aspects affecting the prognosis of patients with HCC. Recent molecular research has identified many tumor biological factors as potential prognostic markers (biomarkers). However, to date, none of them has been proved to be specific enough, and most of the studies for specific molecular parameters were correlative and retrospective. Methodologies, sample sizes, and definitions differ. Consideration should be given to the design of prospective clinical trials in evaluating the prognostic significance of these markers.

These biomarkers could be detected both in tissue and body fluids (serum, urine, bile, etc.). Body fluid (particularly blood and urinary) testing is easily accessible and useful in clinical patients, and is more important in "predicting" the possibility or "early diagnosis" of recurrence and metastasis. So, future work should be focused on serum or urinary markers.

The prognostic significance of circulating DNA in plasma or sesum, and its genetic alterations in HCC, are important trends that deserve attention. Proteomics and cDNA array provide other ways to explore new prognostic markers. So, we can believe, with the continuing progress of human genome project, the development of new molecular and cytogenetic techniques, and a more complete understanding of tumor biology, more and more new prognostic markers with high sensitivity and specificity will soon be found and used in clinical assays. However, the combination of some items, i.e., pathological features and some biomarkers mention ed above, seems to be more practical now.