Review
Copyright ©The Author(s) 2002. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jun 15, 2002; 8(3): 385-392
Published online Jun 15, 2002. doi: 10.3748/wjg.v8.i3.385
The prognostic molecular markers in hepatocellular carcinoma
Lun-Xiu Qin, Zhao-You Tang
Lun-Xiu Qin, Zhao-You Tang, Liver Cancer Institute and Zhongshan Hospital, Fudan University, Shanghai, China
Author contributions: All authors contributed equally to the work.
Correspondence to: Zhao-You Tang, MD, Professor of Surgery and Chairman, Liver Cancer Institute and Zhongshan Hospital, Fudan University, 136 Yi Xue Yuan Road, Shanghai 200032, China. zytang@srcap.stc.sh.cn
Telephone: +86-21-64037181 Fax: +86-21-64037181
Received: March 19, 2002
Revised: April 23, 2002
Accepted: May 8, 2002
Published online: June 15, 2002

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.




INTRODUCTION

Liver cancer is one of the common malignancies worldwide, and has been ranked the 2nd 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.

Footnotes

Edited by Pagliarini R

References
1.  Tang ZY. Hepatocellular carcinoma--cause, treatment and metastasis. World J Gastroenterol. 2001;7:445-454.  [PubMed]  [DOI]
2.  Qin LX, Tang ZY. The prognostic significance of clinical and pathological features in hepatocellular carcinoma. World J Gastroenterol. 2002;8:193-199.  [PubMed]  [DOI]
3.  Zhao WH, Ma ZM, Zhou XR, Feng YZ, Fang BS. Prediction of recurrence and prognosis in patients with hepatocellular carcinoma after resection by use of CLIP score. World J Gastroenterol. 2002;8:237-242.  [PubMed]  [DOI]
4.  Zheng N, Ye SL, Sun RX, Zhao Y, Tang ZY. Effects of cryopreservation and phenylacetate on biological characters of adherent LAK cells from patients with hepatocellular carcinoma. World J Gastroenterol. 2002;8:233-236.  [PubMed]  [DOI]
5.  Korn WM. Moving toward an understanding of the metastatic process in hepatocellular carcinoma. World J Gastroenterol. 2001;7:777-778.  [PubMed]  [DOI]
6.  Mise K, Tashiro S, Yogita S, Wada D, Harada M, Fukuda Y, Miyake H, Isikawa M, Izumi K, Sano N. Assessment of the biological malignancy of hepatocellular carcinoma: relationship to clinicopathological factors and prognosis. Clin Cancer Res. 1998;4:1475-1482.  [PubMed]  [DOI]
7.  Nolte M, Werner M, Nasarek A, Bektas H, von Wasielewski R, Klempnauer J, Georgii A. Expression of proliferation associated antigens and detection of numerical chromosome aberrations in primary human liver tumours: relevance to tumour characteristics and prognosis. J Clin Pathol. 1998;51:47-51.  [PubMed]  [DOI]
8.  Weber JC, Nakano H, Bachellier P, Oussoultzoglou E, Inoue K, Shimura H, Wolf P, Chenard-Neu MP, Jaeck D. Is a proliferation index of cancer cells a reliable prognostic factor after hepatectomy in patients with colorectal liver metastases? Am J Surg. 2001;182:81-88.  [PubMed]  [DOI]
9.  Lin GY, Chen ZL, Lu CM, Li Y, Ping XJ, Huang R. Immunohistochemical study on p53, H-rasp21, c-erbB-2 protein and PCNA expression in HCC tissues of Han and minority ethnic patients. World J Gastroenterol. 2000;6:234-238.  [PubMed]  [DOI]
10.  Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol. 2000;182:311-322.  [PubMed]  [DOI]
11.  Hernández-Rodríguez NA, Correa E, Sotelo R, Contreras-Paredes A, Gomez-Ruiz C, Green L, Mohar A. Ki-67: a proliferative marker that may predict pulmonary metastases and mortality of primary osteosarcoma. Cancer Detect Prev. 2001;25:210-215.  [PubMed]  [DOI]
12.  Ito Y, Matsuura N, Sakon M, Takeda T, Umeshita K, Nagano H, Nakamori S, Dono K, Tsujimoto M, Nakahara M. Both cell proliferation and apoptosis significantly predict shortened disease-free survival in hepatocellular carcinoma. Br J Cancer. 1999;81:747-751.  [PubMed]  [DOI]
13.  Ouchi K, Sugawara T, Ono H, Fujiya T, Kamiyama Y, Kakugawa Y, Mikuni J, Yamanami H, Komatsu S, Horikoshi A. Mitotic index is the best predictive factor for survival of patients with resected hepatocellular carcinoma. Dig Surg. 2000;17:42-48.  [PubMed]  [DOI]
14.  Wellmann A, Flemming P, Behrens P, Wuppermann K, Lang H, Oldhafer K, Pastan I, Brinkmann U. High expression of the proliferation and apoptosis associated CSE1L/CAS gene in hepatitis and liver neoplasms: correlation with tumor progression. Int J Mol Med. 2001;7:489-494.  [PubMed]  [DOI]
15.  Ikeguchi M, Sato N, Hirooka Y, Kaibara N. Computerized nuclear morphometry of hepatocellular carcinoma and its relation to proliferative activity. J Surg Oncol. 1998;68:225-230.  [PubMed]  [DOI]
16.  Veltri RW, Partin AW, Miller MC. Quantitative nuclear grade (QNG): a new image analysis-based biomarker of clinically relevant nuclear structure alterations. J Cell Biochem Suppl. 2000;Suppl 35:151-157.  [PubMed]  [DOI]
17.  Overgaard J, Yilmaz M, Guldberg P, Hansen LL, Alsner J. TP53 mutation is an independent prognostic marker for poor outcome in both node-negative and node-positive breast cancer. Acta Oncol. 2000;39:327-333.  [PubMed]  [DOI]
18.  Takahashi M, Tonoki H, Tada M, Kashiwazaki H, Furuuchi K, Hamada J, Fujioka Y, Sato Y, Takahashi H, Todo S. Distinct prognostic values of p53 mutations and loss of estrogen receptor and their cumulative effect in primary breast cancers. Int J Cancer. 2000;89:92-99.  [PubMed]  [DOI]
19.  Blaszyk H, Hartmann A, Cunningham JM, Schaid D, Wold LE, Kovach JS, Sommer SS. A prospective trial of midwest breast cancer patients: a p53 gene mutation is the most important predictor of adverse outcome. Int J Cancer. 2000;89:32-38.  [PubMed]  [DOI]
20.  Kahlenberg MS, Stoler DL, Rodriguez-Bigas MA, Weber TK, Driscoll DL, Anderson GR, Petrelli NJ. p53 tumor suppressor gene mutations predict decreased survival of patients with sporadic colorectal carcinoma. Cancer. 2000;88:1814-1819.  [PubMed]  [DOI]
21.  Ireland AP, Shibata DK, Chandrasoma P, Lord RV, Peters JH, DeMeester TR. Clinical significance of p53 mutations in adenocarcinoma of the esophagus and cardia. Ann Surg. 2000;231:179-187.  [PubMed]  [DOI]
22.  Tamás L, Kraxner H, Mechtler L, Répássy G, Ribári O, Hirschberg A, Szentkúti G, Járay B, Szentirmay Z. Prognostic significance of P53 histochemistry and DNA histogram parameters in head and neck malignancies. Anticancer Res. 2000;20:4031-4037.  [PubMed]  [DOI]
23.  Murakami I, Hiyama K, Ishioka S, Yamakido M, Kasagi F, Yokosaki Y. p53 gene mutations are associated with shortened survival in patients with advanced non-small cell lung cancer: an analysis of medically managed patients. Clin Cancer Res. 2000;6:526-530.  [PubMed]  [DOI]
24.  Mitsudomi T, Hamajima N, Ogawa M, Takahashi T. Prognostic significance of p53 alterations in patients with non-small cell lung cancer: a meta-analysis. Clin Cancer Res. 2000;6:4055-4063.  [PubMed]  [DOI]
25.  Shahin MS, Hughes JH, Sood AK, Buller RE. The prognostic significance of p53 tumor suppressor gene alterations in ovarian carcinoma. Cancer. 2000;89:2006-2017.  [PubMed]  [DOI]
26.  de Alava E, Antonescu CR, Panizo A, Leung D, Meyers PA, Huvos AG, Pardo-Mindán FJ, Healey JH, Ladanyi M. Prognostic impact of P53 status in Ewing sarcoma. Cancer. 2000;89:783-792.  [PubMed]  [DOI]
27.  Leibovich BC, Cheng L, Weaver AL, Myers RP, Bostwick DG. Outcome prediction with p53 immunostaining after radical prostatectomy in patients with locally advanced prostate cancer. J Urol. 2000;163:1756-1760.  [PubMed]  [DOI]
28.  Osaki T, Kimura T, Tatemoto Y, Dapeng L, Yoneda K, Yamamoto T. Diffuse mode of tumor cell invasion and expression of mutant p53 protein but not of p21 protein are correlated with treatment failure in oral carcinomas and their metastatic foci. Oncology. 2000;59:36-43.  [PubMed]  [DOI]
29.  Suzuki M, Ohwada M, Saga Y, Kohno T, Takei Y, Sato I. Micrometastatic p53-positive cells in the lymph nodes of early stage epithelial ovarian cancer: prognostic significance. Oncology. 2001;60:170-175.  [PubMed]  [DOI]
30.  Shiota G, Ishida M, Noguchi N, Oyama K, Takano Y, Okubo M, Katayama S, Tomie Y, Harada K, Hori K. Circulating p53 antibody in patients with colorectal cancer: relation to clinicopathologic features and survival. Dig Dis Sci. 2000;45:122-128.  [PubMed]  [DOI]
31.  Shao ZM, Wu J, Shen ZZ, Nguyen M. p53 mutation in plasma DNA and its prognostic value in breast cancer patients. Clin Cancer Res. 2001;7:2222-2227.  [PubMed]  [DOI]
32.  Kantola S, Parikka M, Jokinen K, Hyrynkangs K, Soini Y, Alho OP, Salo T. Prognostic factors in tongue cancer - relative importance of demographic, clinical and histopathological factors. Br J Cancer. 2000;83:614-619.  [PubMed]  [DOI]
33.  Ferrero JM, Ramaioli A, Formento JL, Francoual M, Etienne MC, Peyrottes I, Ettore F, Leblanc-Talent P, Namer M, Milano G. P53 determination alongside classical prognostic factors in node-negative breast cancer: an evaluation at more than 10-year follow-up. Ann Oncol. 2000;11:393-397.  [PubMed]  [DOI]
34.  Reed W, Hannisdal E, Boehler PJ, Gundersen S, Host H, Marthin J. The prognostic value of p53 and c-erb B-2 immunostaining is overrated for patients with lymph node negative breast carcinoma: a multivariate analysis of prognostic factors in 613 patients with a follow-up of 14-30 years. Cancer. 2000;88:804-813.  [PubMed]  [DOI]
35.  Kaye PV, Radebold K, Isaacs S, Dent DM. Expression of p53 and p21waf1/cip1 in gastric carcinoma: lack of inter-relationship or correlation with prognosis. Eur J Surg Oncol. 2000;26:39-43.  [PubMed]  [DOI]
36.  Schiller JH, Adak S, Feins RH, Keller SM, Fry WA, Livingston RB, Hammond ME, Wolf B, Sabatini L, Jett J. Lack of prognostic significance of p53 and K-ras mutations in primary resected non-small-cell lung cancer on E4592: a Laboratory Ancillary Study on an Eastern Cooperative Oncology Group Prospective Randomized Trial of Postoperative Adjuvant Therapy. J Clin Oncol. 2001;19:448-457.  [PubMed]  [DOI]
37.  Gadducci A, Cianci C, Cosio S, Carnino F, Fanucchi A, Buttitta F, Conte PF, Genazzani AR. p53 status is neither a predictive nor a prognostic variable in patients with advanced ovarian cancer treated with a paclitaxel-based regimen. Anticancer Res. 2000;20:4793-4799.  [PubMed]  [DOI]
38.  Fleshner N, Kapusta L, Ezer D, Herschorn S, Klotz L. p53 nuclear accumulation is not associated with decreased disease-free survival in patients with node positive transitional cell carcinoma of the bladder. J Urol. 2000;164:1177-1182.  [PubMed]  [DOI]
39.  Gallego MG, Aceñero MJ, Ortega S, Delgado AA, Cantero JL. Prognostic influence of p53 nuclear overexpression in colorectal carcinoma. Dis Colon Rectum. 2000;43:971-975.  [PubMed]  [DOI]
40.  Nieder C, Petersen S, Petersen C, Thames HD. The challenge of p53 as prognostic and predictive factor in Hodgkin's or non-Hodgkin's lymphoma. Ann Hematol. 2001;80:2-8.  [PubMed]  [DOI]
41.  Itoh T, Shiro T, Seki T, Nakagawa T, Wakabayashi M, Inoue K, Okamura A. Relationship between p53 overexpression and the proliferative activity in hepatocellular carcinoma. Int J Mol Med. 2000;6:137-142.  [PubMed]  [DOI]
42.  Jeng KS, Sheen IS, Chen BF, Wu JY. Is the p53 gene mutation of prognostic value in hepatocellular carcinoma after resection? Arch Surg. 2000;135:1329-1333.  [PubMed]  [DOI]
43.  Tang Z, Qin L, Wang X, Zhou G, Liao Y, Weng Y, Jiang X, Lin Z, Liu K, Ye S. Alterations of oncogenes, tumor suppressor genes and growth factors in hepatocellular carcinoma: with relation to tumor size and invasiveness. Chin Med J (Engl). 1998;111:313-318.  [PubMed]  [DOI]
44.  Sugo H, Takamori S, Kojima K, Beppu T, Futagawa S. The significance of p53 mutations as an indicator of the biological behavior of recurrent hepatocellular carcinomas. Surg Today. 1999;29:849-855.  [PubMed]  [DOI]
45.  Heinze T, Jonas S, Kärsten A, Neuhaus P. Determination of the oncogenes p53 and C-erb B2 in the tumour cytosols of advanced hepatocellular carcinoma (HCC) and correlation to survival time. Anticancer Res. 1999;19:2501-2503.  [PubMed]  [DOI]
46.  Qin LX, Tang ZY, Ma ZC, Wu ZQ, Zhou XD, Ye QH, Ji Y, Huang LW, Jia HL, Sun HC. P53 immunohistochemistry scoring is an independent prognostic marker of patient with hepatocellular carcinoma resection: A prospective study of 256 formalin-fixed paraffin-embedded tumor samples. World J Gastroenterol. 2002;(in press).  [PubMed]  [DOI]
47.  Shiota G, Kishimoto Y, Suyama A, Okubo M, Katayama S, Harada K, Ishida M, Hori K, Suou T, Kawasaki H. Prognostic significance of serum anti-p53 antibody in patients with hepatocellular carcinoma. J Hepatol. 1997;27:661-668.  [PubMed]  [DOI]
48.  Tangkijvanich P, Janchai A, Charuruks N, Kullavanijaya P, Theamboonlers A, Hirsch P, Poovorawan Y. Clinical associations and prognostic significance of serum anti-p53 antibodies in Thai patients with hepatocellular carcinoma. Asian Pac J Allergy Immunol. 2000;18:237-243.  [PubMed]  [DOI]
49.  Saffroy R, Lelong JC, Azoulay D, Salvucci M, Reynes M, Bismuth H, Debuire B, Lemoine A. Clinical significance of circulating anti-p53 antibodies in European patients with hepatocellular carcinoma. Br J Cancer. 1999;79:604-610.  [PubMed]  [DOI]
50.  Endo K, Ueda T, Ohta T, Terada T. Protein expression of MDM2 and its clinicopathological relationships in human hepatocellular carcinoma. Liver. 2000;20:209-215.  [PubMed]  [DOI]
51.  Chao Y, Shih YL, Chiu JH, Chau GY, Lui WY, Yang WK, Lee SD, Huang TS. Overexpression of cyclin A but not Skp 2 correlates with the tumor relapse of human hepatocellular carcinoma. Cancer Res. 1998;58:985-990.  [PubMed]  [DOI]
52.  Ohashi R, Gao C, Miyazaki M, Hamazaki K, Tsuji T, Inoue Y, Uemura T, Hirai R, Shimizu N, Namba M. Enhanced expression of cyclin E and cyclin A in human hepatocellular carcinomas. Anticancer Res. 2001;21:657-662.  [PubMed]  [DOI]
53.  Ito Y, Takeda T, Sakon M, Monden M, Tsujimoto M, Matsuura N. Expression and prognostic role of cyclin-dependent kinase 1 (cdc2) in hepatocellular carcinoma. Oncology. 2000;59:68-74.  [PubMed]  [DOI]
54.  Philipp-Staheli J, Payne SR, Kemp CJ. p27(Kip1): regulation and function of a haploinsufficient tumor suppressor and its misregulation in cancer. Exp Cell Res. 2001;264:148-168.  [PubMed]  [DOI]
55.  Fiorentino M, Altimari A, D'Errico A, Cukor B, Barozzi C, Loda M, Grigioni WF. Acquired expression of p27 is a favorable prognostic indicator in patients with hepatocellular carcinoma. Clin Cancer Res. 2000;6:3966-3972.  [PubMed]  [DOI]
56.  Ito Y, Matsuura N, Sakon M, Miyoshi E, Noda K, Takeda T, Umeshita K, Nagano H, Nakamori S, Dono K. Expression and prognostic roles of the G1-S modulators in hepatocellular carcinoma: p27 independently predicts the recurrence. Hepatology. 1999;30:90-99.  [PubMed]  [DOI]
57.  Tannapfel A, Wasner M, Krause K, Geissler F, Katalinic A, Hauss J, Mössner J, Engeland K, Wittekind C. Expression of p73 and its relation to histopathology and prognosis in hepatocellular carcinoma. J Natl Cancer Inst. 1999;91:1154-1158.  [PubMed]  [DOI]
58.  Qin Y, Li B, Tan YS, Sun ZL, Zuo FQ, Sun ZF. Polymorphism of p16INK4a gene and rare mutation of p15INK4b gene exon2 in primary hepatocarcinoma. World J Gastroenterol. 2000;6:411-414.  [PubMed]  [DOI]
59.  Wang Q, Lin ZY, Feng XL. Alterations in metastatic properties of hepatocellular carcinoma cell following H-ras oncogene transfection. World J Gastroenterol. 2001;7:335-339.  [PubMed]  [DOI]
60.  Cui J, Yang DH, Bi XJ, Fan ZR. Methylation status of c-fms oncogene in HCC and its relationship with clinical pathology. World J Gastroenterol. 2001;7:136-139.  [PubMed]  [DOI]
61.  Kawate S, Fukusato T, Ohwada S, Watanuki A, Morishita Y. Amplification of c-myc in hepatocellular carcinoma: correlation with clinicopathologic features, proliferative activity and p53 overexpression. Oncology. 1999;57:157-163.  [PubMed]  [DOI]
62.  Tavian D, De Petro G, Benetti A, Portolani N, Giulini SM, Barlati S. u-PA and c-MET mRNA expression is co-ordinately enhanced while hepatocyte growth factor mRNA is down-regulated in human hepatocellular carcinoma. Int J Cancer. 2000;87:644-649.  [PubMed]  [DOI]
63.  Luo YQ, Wu MC, Cong WM. Gene expression of hepatocyte growth factor and its receptor in HCC and nontumorous liver tissues. World J Gastroenterol. 1999;5:119-121.  [PubMed]  [DOI]
64.  Prange W, Schirmacher P. Absence of therapeutically relevant c-erbB-2 expression in human hepatocellular carcinomas. Oncol Rep. 2001;8:727-730.  [PubMed]  [DOI]
65.  Ito Y, Takeda T, Sakon M, Tsujimoto M, Higashiyama S, Noda K, Miyoshi E, Monden M, Matsuura N. Expression and clinical significance of erb-B receptor family in hepatocellular carcinoma. Br J Cancer. 2001;84:1377-1383.  [PubMed]  [DOI]
66.  Ito Y, Miyoshi E, Takeda T, Sakon M, Noda K, Tsujimoto M, Monden M, Taniguchi N, Matsuura N. Expression and possible role of ets-1 in hepatocellular carcinoma. Am J Clin Pathol. 2000;114:719-725.  [PubMed]  [DOI]
67.  Ito Y, Monden M, Takeda T, Eguchi H, Umeshita K, Nagano H, Nakamori S, Dono K, Sakon M, Nakamura M. The status of Fas and Fas ligand expression can predict recurrence of hepatocellular carcinoma. Br J Cancer. 2000;82:1211-1217.  [PubMed]  [DOI]
68.  Wang XZ, Chen XC, Yang YH, Chen ZX, Huang YH, Tao QM. Relationship between HBxAg and Fas/FasL in patients with hepatocellular carcinoma. World J Gas troenterol. 2000;6:S17.  [PubMed]  [DOI]
69.  Kobayashi T, Kubota K, Takayama T, Makuuchi M. Telomerase activity as a predictive marker for recurrence of hepatocellular carcinoma after hepatectomy. Am J Surg. 2001;181:284-288.  [PubMed]  [DOI]
70.  Tatsuma T, Goto S, Kitano S, Lin YC, Lee CM, Chen CL. Telomerase activity in peripheral blood for diagnosis of hepatoma. J Gastroenterol Hepatol. 2000;15:1064-1070.  [PubMed]  [DOI]
71.  Suda T, Isokawa O, Aoyagi Y, Nomoto M, Tsukada K, Shimizu T, Suzuki Y, Naito A, Igarashi H, Yanagi M. Quantitation of telomerase activity in hepatocellular carcinoma: a possible aid for a prediction of recurrent diseases in the remnant liver. Hepatology. 1998;27:402-406.  [PubMed]  [DOI]
72.  Huang GT, Lee HS, Chen CH, Sheu JC, Chiou LL, Chen DS. Correlation of E-cadherin expression and recurrence of hepatocellular carcinoma. Hepatogastroenterology. 1999;46:1923-1927.  [PubMed]  [DOI]
73.  Endo K, Ueda T, Ueyama J, Ohta T, Terada T. Immunoreactive E-cadherin, alpha-catenin, beta-catenin, and gamma-catenin proteins in hepatocellular carcinoma: relationships with tumor grade, clinicopathologic parameters, and patients' survival. Hum Pathol. 2000;31:558-565.  [PubMed]  [DOI]
74.  Wong CM, Fan ST, Ng IO. beta-Catenin mutation and overexpression in hepatocellular carcinoma: clinicopathologic and prognostic significance. Cancer. 2001;92:136-145.  [PubMed]  [DOI]
75.  Cui J, Zhou XD, Liu YK, Tang ZY, Zile MH. Abnormal beta-catenin gene expression with invasiveness of primary hepatocellular carcinoma in China. World J Gastroenterol. 2001;7:542-546.  [PubMed]  [DOI]
76.  Hsu HC, Jeng YM, Mao TL, Chu JS, Lai PL, Peng SY. Beta-catenin mutations are associated with a subset of low-stage hepatocellular carcinoma negative for hepatitis B virus and with favorable prognosis. Am J Pathol. 2000;157:763-770.  [PubMed]  [DOI]
77.  Sun JJ, Zhou XD, Liu YK, Tang ZY, Feng JX, Zhou G, Xue Q, Chen J. Invasion and metastasis of liver cancer: expression of intercellular adhesion molecule 1. J Cancer Res Clin Oncol. 1999;125:28-34.  [PubMed]  [DOI]
78.  Mei MH, Xu J, Shi QF, Yang JH, Chen Q, Qin LL. Clinical significance of serum intercellular adhesion molecule-1 detection in patients with hepatocellular carcinoma. World J Gastroenterol. 2000;6:408-410.  [PubMed]  [DOI]
79.  Xu J, Mei MH, Zeng SE, Shi QF, Liu YM, Qin LL. Expressions of ICAM-1 and its mRNA in sera and tissues of patients with hepatocellular carcinoma. World J Gastroenterol. 2001;7:120-125.  [PubMed]  [DOI]
80.  Goodison S, Urquidi V, Tarin D. CD44 cell adhesion molecules. Mol Pathol. 1999;52:189-196.  [PubMed]  [DOI]
81.  Endo K, Terada T. Protein expression of CD44 (standard and variant isoforms) in hepatocellular carcinoma: relationships with tumor grade, clinicopathologic parameters, p53 expression, and patient survival. J Hepatol. 2000;32:78-84.  [PubMed]  [DOI]
82.  Sakamoto Y, Mafune K, Mori M, Shiraishi T, Imamura H, Mori M, Takayama T, Makuuchi M. Overexpression of MMP-9 correlates with growth of small hepatocellular carcinoma. Int J Oncol. 2000;17:237-243.  [PubMed]  [DOI]
83.  Jiang YF, Yang ZH, Hu JQ. Recurrence or metastasis of HCC: predictors, early detection and experimental antiangiogenic therapy. World J Gastroenterol. 2000;6:61-65.  [PubMed]  [DOI]
84.  Bu W, Tang ZY, Ye SL, Liu KD, Huang XW, Gao DM. The association of type IV collagenase with invasion and metasta-sis of hepatocellular carcinoma. Zhonghua Xiaohua Zazhi. 1999;19:13-15.  [PubMed]  [DOI]
85.  Fox SB, Taylor M, Grøndahl-Hansen J, Kakolyris S, Gatter KC, Harris AL. Plasminogen activator inhibitor-1 as a measure of vascular remodelling in breast cancer. J Pathol. 2001;195:236-243.  [PubMed]  [DOI]
86.  Zheng Q, Tang ZY, Xue Q, Shi DR, Song HY, Tang HB. Invasion and metastasis of hepatocellular carcinoma in relation to urokinase-type plasminogen activator, its receptor and inhibitor. J Cancer Res Clin Oncol. 2000;126:641-646.  [PubMed]  [DOI]
87.  Itoh T, Hayashi Y, Kanamaru T, Morita Y, Suzuki S, Wang W, Zhou L, Rui JA, Yamamoto M, Kuroda Y. Clinical significance of urokinase-type plasminogen activator activity in hepatocellular carcinoma. J Gastroenterol Hepatol. 2000;15:422-430.  [PubMed]  [DOI]
88.  Kuroi K, Toi M. Circulating angiogenesis regulators in cancer patients. Int J Biol Markers. 2001;16:5-26.  [PubMed]  [DOI]
89.  Morinaga S, Imada T, Shimizu A, Akaike M, Sugimasa Y, Takemiya S, Takanashi Y. Angiogenesis in hepatocellular carcinoma as evaluated by alpha smooth muscle actin immunohistochemistry. Hepatogastroenterology. 2001;48:224-228.  [PubMed]  [DOI]
90.  El-Assal ON, Yamanoi A, Soda Y, Yamaguchi M, Igarashi M, Yamamoto A, Nabika T, Nagasue N. Clinical significance of microvessel density and vascular endothelial growth factor expression in hepatocellular carcinoma and surrounding liver: possible involvement of vascular endothelial growth factor in the angiogenesis of cirrhotic liver. Hepatology. 1998;27:1554-1562.  [PubMed]  [DOI]
91.  Sun HC, Tang ZY, Li XM, Zhou YN, Sun BR, Ma ZC. Microvessel density of hepatocellular carcinoma: its relationship with prognosis. J Cancer Res Clin Oncol. 1999;125:419-426.  [PubMed]  [DOI]
92.  Poon RT, Fan ST, Wong J. Clinical implications of circulating angiogenic factors in cancer patients. J Clin Oncol. 2001;19:1207-1225.  [PubMed]  [DOI]
93.  Poon RT, Ng IO, Lau C, Zhu LX, Yu WC, Lo CM, Fan ST, Wong J. Serum vascular endothelial growth factor predicts venous invasion in hepatocellular carcinoma: a prospective study. Ann Surg. 2001;233:227-235.  [PubMed]  [DOI]
94.  Li XM, Tang ZY, Qin LX, Zhou J, Sun HC. Serum vascular endothelial growth factor is a predictor of invasion and metastasis in hepatocellular carcinoma. J Exp Clin Cancer Res. 1999;18:511-517.  [PubMed]  [DOI]
95.  Niu Q, Tang ZY, Ma ZC, Qin LX, Zhang LH. Serum vascular endothelial growth factor is a potential biomarker of metastatic recurrence after curative resection of hepatocellular carcinoma. World J Gastroenterol. 2000;6:565-568.  [PubMed]  [DOI]
96.  Zhou J, Tang ZY, Fan J, Wu ZQ, Li XM, Liu YK, Liu F, Sun HC, Ye SL. Expression of platelet-derived endothelial cell growth factor and vascular endothelial growth factor in hepatocellular carcinoma and portal vein tumor thrombus. J Cancer Res Clin Oncol. 2000;126:57-61.  [PubMed]  [DOI]
97.  Tada K, Shiraishi S, Kamiryo T, Nakamura H, Hirano H, Kuratsu J, Kochi M, Saya H, Ushio Y. Analysis of loss of heterozygosity on chromosome 10 in patients with malignant astrocytic tumors: correlation with patient age and survival. J Neurosurg. 2001;95:651-659.  [PubMed]  [DOI]
98.  Bisgaard ML, Jäger AC, Dalgaard P, Søndergaard JO, Rehfeld JF, Nielsen FC. Allelic loss of chromosome 2p21-16.3 is associated with reduced survival in sporadic colorectal cancer. Scand J Gastroenterol. 2001;36:405-409.  [PubMed]  [DOI]
99.  Hirano A, Emi M, Tsuneizumi M, Utada Y, Yoshimoto M, Kasumi F, Akiyama F, Sakamoto G, Haga S, Kajiwara T. Allelic losses of loci at 3p25.1, 8p22, 13q12, 17p13.3, and 22q13 correlate with postoperative recurrence in breast cancer. Clin Cancer Res. 2001;7:876-882.  [PubMed]  [DOI]
100.  Simoneau M, LaRue H, Aboulkassim TO, Meyer F, Moore L, Fradet Y. Chromosome 9 deletions and recurrence of superficial bladder cancer: identification of four regions of prognostic interest. Oncogene. 2000;19:6317-6323.  [PubMed]  [DOI]
101.  Washburn JG, Wojno KJ, Dey J, Powell IJ, Macoska JA. 8pter-p23 deletion is associated with racial differences in prostate cancer outcome. Clin Cancer Res. 2000;6:4647-4652.  [PubMed]  [DOI]
102.  Wong N, Lai P, Lee SW, Fan S, Pang E, Liew CT, Sheng Z, Lau JW, Johnson PJ. Assessment of genetic changes in hepatocellular carcinoma by comparative genomic hybridization analysis: relationship to disease stage, tumor size, and cirrhosis. Am J Pathol. 1999;154:37-43.  [PubMed]  [DOI]
103.  Itano O, Ueda M, Kikuchi K, Shimazu M, Kitagawa Y, Aiura K, Kitajima M. A new predictive factor for hepatocellular carcinoma based on two-dimensional electrophoresis of genomic DNA. Oncogene. 2000;19:1676-1683.  [PubMed]  [DOI]
104.  Qin LX, Tang ZY, Sham JS, Ma ZC, Ye SL, Zhou XD, Wu ZQ, Trent JM, Guan XY. The association of chromosome 8p deletion and tumor metastasis in human hepatocellular carcinoma. Cancer Res. 1999;59:5662-5665.  [PubMed]  [DOI]
105.  Qin LX, Tang ZY, Ye SL, Liu YK, Ma ZC, Zhou XD, Wu ZQ, Lin ZY, Sun FX, Tian J. Chromosome 8p deletion is associated with metastasis of human hepatocellular carcinoma when high and low metastatic models are compared. J Cancer Res Clin Oncol. 2001;127:482-488.  [PubMed]  [DOI]
106.  Zhang LH, Qin LX, Ma ZC, Ye SL, Liu YK, Ye QH, Wu X, Huang W, Tang ZY. Allelic imbalance regions on chromosomes 8p, 17p and 19p related to metastasis of hepatocellular carcinoma: comparison between matched primary and metastatic lesions in 22 patients by genome-wide microsatellite analysis. J Cancer Res Clin Oncol. 2003;129:279-286.  [PubMed]  [DOI]
107.  Anker P, Stroun M. Circulating DNA in plasma or serum. Medicina (B Aires). 2000;60:699-702.  [PubMed]  [DOI]
108.  Taback B, Fujiwara Y, Wang HJ, Foshag LJ, Morton DL, Hoon DS. Prognostic significance of circulating microsatellite markers in the plasma of melanoma patients. Cancer Res. 2001;61:5723-5726.  [PubMed]  [DOI]
109.  Nunes DN, Kowalski LP, Simpson AJ. Circulating tumor-derived DNA may permit the early diagnosis of head and neck squamous cell carcinomas. Int J Cancer. 2001;92:214-219.  [PubMed]  [DOI]
110.  Kennedy S. Proteomic profiling from human samples: the body fluid alternative. Toxicol Lett. 2001;120:379-384.  [PubMed]  [DOI]
111.  Tsai JF, Chuang LY, Jeng JE, Yang ML, Chang WY, Hsieh MY, Lin ZY, Tsai JH. Clinical relevance of transforming growth factor-beta 1 in the urine of patients with hepatocellular carcinoma. Medicine (Baltimore). 1997;76:213-226.  [PubMed]  [DOI]
112.  King KL, Li AF, Chau GY, Chi CW, Wu CW, Huang CL, Lui WY. Prognostic significance of heat shock protein-27 expression in hepatocellular carcinoma and its relation to histologic grading and survival. Cancer. 2000;88:2464-2470.  [PubMed]  [DOI]
113.  Osada T, Nagashima I, Tsuno NH, Kitayama J, Nagawa H. Prognostic significance of glutamine synthetase expression in unifocal advanced hepatocellular carcinoma. J Hepatol. 2000;33:247-253.  [PubMed]  [DOI]
114.  Kondo M, Yamamoto H, Nagano H, Okami J, Ito Y, Shimizu J, Eguchi H, Miyamoto A, Dono K, Umeshita K. Increased expression of COX-2 in nontumor liver tissue is associated with shorter disease-free survival in patients with hepatocellular carcinoma. Clin Cancer Res. 1999;5:4005-4012.  [PubMed]  [DOI]
115.  Chau GY, Wu CW, Lui WY, Chang TJ, Kao HL, Wu LH, King KL, Loong CC, Hsia CY, Chi CW. Serum interleukin-10 but not interleukin-6 is related to clinical outcome in patients with resectable hepatocellular carcinoma. Ann Surg. 2000;231:552-558.  [PubMed]  [DOI]
116.  Pirisi M, Toniutto P, Uzzau A, Fabris C, Avellini C, Scott C, Apollonio L, Beltrami CA, Bresadola F. Carriage of HFE mutations and outcome of surgical resection for hepatocellular carcinoma in cirrhotic patients. Cancer. 2000;89:297-302.  [PubMed]  [DOI]
117.  Yamamoto Y, Sakamoto M, Fujii G, Kanetaka K, Asaka M, Hirohashi S. Cloning and characterization of a novel gene, DRH1, down-regulated in advanced human hepatocellular carcinoma. Clin Cancer Res. 2001;7:297-303.  [PubMed]  [DOI]
118.  Furumoto K, Arii S, Mori A, Furuyama H, Gorrin Rivas MJ, Nakao T, Isobe N, Murata T, Takahashi C, Noda M. RECK gene expression in hepatocellular carcinoma: correlation with invasion-related clinicopathological factors and its clinical significance. Reverse-inducing--cysteine-rich protein with Kazal motifs. Hepatology. 2001;33:189-195.  [PubMed]  [DOI]
119.  Ye QH, Qin LX, Forgues M, He P, Kim JW, Peng AC, Simon R, Robles A, C hen YD, Ma ZC. Gene expression profiling and supervised machine learning to define metastasis-related genes in human he patocellular carcinoma. Nature Med. 2002;(submitted).  [PubMed]  [DOI]