10.1245/ASO.2006.07.018
Annals of Surgical Oncology 13:947-954 (2006)
© 2006 Society of Surgical Oncology
Specific Gene-Expression Profiles of Noncancerous Liver Tissue Predict the Risk for Multicentric Occurrence of Hepatocellular Carcinoma in Hepatitis C Virus–Positive Patients
Masahiro Okamoto, MD1,
Tohru Utsunomiya, MD1,
Shigeki Wakiyama, MD2,
Masaji Hashimoto, MD3,
Kengo Fukuzawa, MD4,
Takahiro Ezaki, MD5,
Taizo Hanai, MD6,
Hiroshi Inoue, MD1 and
Masaki Mori, MD1
1 Department of Molecular and Surgical Oncology, Medical Institute of Bioregulation, Kyushu University, Tsurumihara 4546, Beppu, 874-0838, Japan
2 Department of Surgery, Iizuka Hospital, Iizuka, Japan
3 Department of Digestive Surgery, Toranomon Hospital and Okinaka Memorial Institute for Medical Research, Tokyo, Japan
4 Department of Surgery, Oita Red Cross Hospital, Oita, Japan
5 Department of Surgery, Hiroshima Red Cross Hospital and Atomic Bomb Survivors Hospital, Hiroshima, Japan
6 Laboratory for Bioinformatics, Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, Japan
Correspondence: Address correspondence and reprint requests to: Masaki Mori, MD; E-mail: mmori{at}beppu.kyushu-u.ac.jp.
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ABSTRACT
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Background: Hepatitis C virus (HCV) infection produces chronic hepatitis, cirrhosis, and, ultimately, hepatocellular carcinoma (HCC). A molecular analysis of the damaged liver tissues infected with HCV may identify specific gene-expression profiles associated with a risk for liver carcinogenesis.
Methods: Forty patients with HCV-positive HCC were classified into two groups: single nodular HCC group (n = 28) and multicentric HCC group (n = 12). Using a complementary DNA microarray, we compared the gene-expression patterns of the noncancerous liver tissue specimens between the two groups. We also identified the differentially expressed genes related to multicentric recurrence in the liver remnant. We then evaluated whether a specific gene-expression profile can accurately estimate the risk for multicentric hepatocarcinogenesis.
Results: We selected the 230 differentially expressed genes in the multicentric HCC group. A hierarchical clustering analysis identified a cluster that might be closely associated with the multicentric occurrence of HCC. On the basis of the gene-expression profiling of the 36 genes commonly associated with both multicentric HCC and multicentric recurrence, we created a scoring system to estimate the risk for multicentric hepatocarcinogenesis. The prediction score of patients in the multicentric HCC group with multicentric recurrence (19.9 ± 9.2) was significantly higher (P < .05) than that in the single nodular HCC group without multicentric recurrence (–1.8 ± 12.7).
Conclusions: Specific gene-expression signatures in noncancerous liver tissue may help to accurately predict the risk for developing HCC.
Key Words: Hepatocellular carcinoma • Multicentric occurrence • Hepatitis C virus • DNA microarray
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INTRODUCTION
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Hepatocellular carcinoma (HCC) is a common malignancy that causes significant morbidity and mortality worldwide,1 and hepatitis C virus (HCV) infection is closely associated with the development of HCC.2 Although the mechanisms of liver carcinogenesis associated with HCV infection are still not well known, the increased turnover of hepatocytes and inflammatory cell infiltrate seen in chronic HCV hepatitis and cirrhosis may lead to an accumulation of genetic alterations that ultimately result in the development of HCC.3,4
The long-term results of hepatic resection for HCC in cirrhotic patients have been disappointing because of the high rate of intrahepatic recurrence as a result of either multicentric occurrence or intrahepatic metastasis of HCC.5,6 The dismal outcomes of HCC patients even after a curative hepatectomy have been, at least partially, due to its multicentric origin.7–9
Several studies have shown that the specific gene-expression patterns in cancerous tissues of HCC could accurately predict early intrahepatic recurrence, possibly because of the intrahepatic metastasis of HCC.10,11 However, to examine the molecular mechanisms during the process of liver carcinogenesis on the basis of the idea of field cancerization, it is thus considered reasonable to investigate the non-cancerous portion of the liver tissue because multicentric occurrence of HCC is mainly associated with underlying chronic liver damage rather than adverse tumor factors.7,12 Because molecular alterations in hepatocarcinogenesis might differ regarding the etiology of chronic liver damage,12 in this study, we focused on examining noncancerous liver tissues obtained from patients with HCV-related HCC. We then attempted to explore a specific gene-expression signature that could enable us to accurately predict the risk for multicentric hepatocarcinogenesis.
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METHODS
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Liver Tissue Samples
Forty patients with HCV-positive HCC and seven patients with colorectal liver metastasis who had undergone an initial hepatectomy at our institute and the affiliated hospitals were entered onto this study. The last seven patients were carefully examined and found to be seronegative for either hepatitis B surface antigen or HCV antibody, and they all had liver function values within the normal limits and had histologically normal livers. The resected noncancerous liver tissue specimens were meticulously taken as far from the tumor as possible and were immediately frozen in liquid nitrogen and kept at –80°C until the following RNA preparation. According to The General Rules for the Clinical and Pathological Study of Primary Liver Cancer (4th edition),13 intrahepatic metastases of HCC were defined as (1) tumors that seemed to have developed from portal tumor emboli, (2) tumors that were distributed in a gradient-like pattern (i.e., clustered more densely around the largest lesion and more sparsely farther from it), and (3) tumors that had separate tumors near the largest lesion that were clearly smaller than the largest lesion and were histologically similar to or less differentiated than the largest one. In this study, patients with intrahepatic metastasis of HCC and those with tumors too difficult to clarify were excluded. We also excluded patients who developed early recurrence within 18 months that was obviously due to the intrahepatic metastases of HCC, as determined by the imaging strategies, including abdominal ultrasonography, computed tomography (CT), CT during arterial portography, and magnetic resonance imaging (MRI).14
Forty samples were classified into two groups on the basis of the histological findings: noncancerous tissue specimens from the patients with single nodular HCC (SN group; n = 28) and those from the patients with multicentric HCC (MC group; n = 12). Written informed consent was obtained from all patients according to the guidelines approved by the institutional research board at each hospital. All patients were closely followed up after surgery at regular 1-month intervals. The mean follow-up time was 33 months (range, 18–41 months). Each follow-up visit included a physical examination, blood chemistry tests, and measurements of the serum levels of alfa fetoprotein and proteins induced by vitamin K absence II. Abdominal ultrasonography, CT, and MRI were all performed at 3- to 4-month intervals. When recurrence was suspected, the patient was readmitted for an angiographic examination.15
RNA Preparation
Total liver RNA was isolated from frozen tissue by a guanidine/cesium trifluoroacetate extraction method by using a Quick Prep total RNA extraction kit (Amersham Pharmacia Biotech, Little Chalfont, UK). To ensure the use of only high-quality RNA, the concentration and purity were determined by an Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA).16
Complementary DNA Microarray (Labeling, Hybridization, and Scanning)
We used the commercially available Agilent Human 1 cDNA Microarray that contains 12,814 clones, listed at http://www.agilent.com/chem/genelists. Twenty-microgram aliquots of each total RNA from the 40 liver tissue specimens infected with HCV were labeled with Cy5-deoxyuridine triphosphate (Amersham Pharmacia Biotech), whereas a 20-µg aliquot of total RNA from a mixture of the 7 normal liver samples was labeled with Cy3-deoxyuridine triphosphate as a control. The labeled probes were hybridized with an Agilent Human 1 cDNA Microarray Kit in hybridization buffer for 17 hours at 65°C. After hybridization, the slides were washed in .5x standard saline citrate and .01% sodium dodecylsulfate for 5 minutes at room temperature and .06x standard saline citrate for 2 minutes at 22°C. The Cy3 and Cy5 fluorescent intensities for each clone were determined by a confocal laser scanner (MAS-O; Fuji Photo Film, Tokyo, Japan) and analyzed by ArrayGauge (version 1.2; Fuji Photo Film) to correct for any background signals. The expression values were calculated as the logarithm of Cy5:Cy3 ratios and normalized by the ArrayGauge software. The expression levels below 1 U (a cutoff value of this scanner) were assigned a value of 1. When the gene was not expressed in Cy5 or Cy3 signals (expression value = 0), the data were defined as missing.17
Identification of Differentially Regulated Genes
We compared the nontumor liver tissue specimens in the MC group (n = 12) with those in the SN group (n = 28) by using Student’s t-test and calculated the signal-to-noise ratio for each gene. The signal-to-noise ratio is calculated as follows:
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where µ and 
represent the mean and standard deviation of expression values for the MC group and SN group, respectively. We considered a gene to be a "multicentric HCC-associated gene" when its P value was <.05. Among the 40 patients with HCC, 15 patients were found to develop multicentric recurrence in their liver remnant during our follow-up period. We then compared the noncancerous liver samples in the patients with recurrence (n = 15) with those in the patients without recurrence in the liver remnant (n = 25) by using Student’s t-test and calculated the P value for each gene. We considered the gene to be a "multicentric recurrence-associated gene" when its P value was <.05.
Development of Prediction Scores for the Risk of Multicentric Hepatocarcinogenesis
Assume that we have m cases whose prediction scores were to be calculated and n genes that were valuable for predicting the multicentric hepatocarcinogenesis. Let Eij be the gene expression value of gene j, for j = 1....n, measured by complementary DNA (cDNA) microarray in case i, for i = 1....m. We first calculated the Sij of gene j in case i as below:
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where Ejmc is the mean expression value of the patients in the MC group and Ejsn is that of the patients in the SN group. If Sij is >1.0, then Sij is assigned a value of 1.0, and if Sij is less than –1.0, then Sij is assigned a value of –1.0. We then calculated the prediction score of the multicentric recurrence of case i (Si):
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where Wj is the signal-to-noise ratio of each gene (j) as calculated according to formula 1. Agglomerative hierarchical clustering18 was applied by using the complete linkage method by Cluster and Tree-view software obtained from http://rana.lbl.gov/EisenSoftware.htm.
Statistical Analysis
For continuous variables, the data were expressed as mean ± SD. We used the 
2 test and Student’s t-test to assess whether clinicopathologic factors were associated with multicentric occurrence of HCC. The survival curves were plotted according to the Kaplan-Meier method, and the generalized Wilcoxon test was applied to compare the survival curve. We used analysis of variance to compare the prediction scores among the groups, and statistical significance was estimated by the Tukey-Kramer honestly significant difference test. The findings were considered to be significant when the P value was <.05.
Additional Microarray Information
This microarray study followed the minimum information about a microarray experiment guidelines issued by the Microarray Gene Expression Data group.19
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RESULTS
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Clinicopathologic Characteristics and Prognosis in Patients With Multicentric HCC
Table 1
summarizes the clinicopathologic variables of the patients in the MC group (n = 12) and the SN group (n = 28). No differences were observed between the two groups regarding the liver function tests and pathologic variables regarding both the tumor factors and nontumorous liver tissues. We compared the disease-free survival curves after a curative hepatectomy between the two groups (Fig. 1). The disease-free survival rate of patients in the MC group was significantly lower than that of the patients in the SN group (P < .05).
Received for publication July 19, 2005.
Accepted for publication January 4, 2006.
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ABSTRACT
INTRODUCTION
). These findings suggest that common features in the gene-expression patterns might exist between synchronous multicentric HCC and metachronous multicentric recurrence. We next selected the differentially expressed genes associated with multicentric recurrence in the liver remnant (527 multicentric recurrence-associated genes). In this study, we histopathologically defined synchronous multicentric HCC according to The General Rules for the Clinical and Pathological Study of Primary Liver Cancer.13 We defined the multicentric recurrence of HCC according to the findings of imaging modalities such as CT, CT during arterial portography, and MRI.14 We initially excluded any patients who developed early recurrences within 18 months that were apparently caused by intrahepatic metastases of HCC. Therefore, the recurrence in the liver remnant detected in the 15 patients were considered to be due to multicentric recurrence, although the mean follow-up time was relatively short. One of these 15 patients who underwent a repeat hepatectomy for a recurrent tumor was histopathologically determined to have multicentric recurrence. The patients diagnosed as having both intrahepatic metastases and multicentric occurrence of HCC were included in the patients with multicentric recurrences because the gene-expression patterns in the noncancerous liver tissues of these patients should theoretically reflect the risk for the multicentric occurrence of HCC. We subsequently identified a common gene-expression signature shared by the multicentric HCC-related genes and the multicentric recurrence-related genes (Fig. 3A
