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© 2003 American Society for Clinical Oncology Elevated Expression of Valosin-Containing Protein (p97) in Hepatocellular Carcinoma Is Correlated With Increased Incidence of Tumor RecurrenceFrom the Departments of Surgery and Clinical Oncology, and Pathology, Osaka University Graduate School of Medicine, Osaka, Japan. Address reprint requests to Yasuhiko Tomita, MD, Department of Pathology (C3), Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita Osaka 565-0871, Japan; email: yt{at}molpath.med.osaka-u.ac.jp.
Purpose: Valosin-containing protein (VCP; also known as p97) has been shown to be associated with antiapoptotic function and metastasis via activation of the nuclear factor-  B signaling pathway. In this study, association of VCP expression with recurrence of hepatocellular carcinoma (HCC) and patient survival was examined. Patients and Methods: VCP expression in 170 patients (139 male and 31 female) with ages ranging from 31 to 81 years (median, 61 years) was analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, in which staining intensity in tumor cells was categorized as weaker (level 1) or equal to or stronger (level 2) than that in endothelial cells. Results: Immunohistochemically, 57 patients (35.2%) showed level 1, and 105 patients (64.8%) showed level 2, VCP expression. Quantitative RT-PCR analysis revealed higher VCP mRNA expression in level 2 patients (n = 7) than level 1 (n = 4) (P < .05). Patients with VCP-level 2 HCC showed higher rate of portal vein invasion in the tumor (P < .01) and poorer disease-free and overall survival (P < .0001 and P < .05, respectively) compared with level 1 patients. Multivariate analysis revealed VCP expression level, tumor multiplicity, and degree of fibrosis in the noncancerous liver tissue to be independent prognosticators for disease-free and overall survival. VCP level was an indicator for disease-free survival in each early- (I and II) and advanced- (III and IV) stage group of pathologic tumor-node-metastasis classification (P < .001 and P < .01, respectively). Conclusion: VCP expression level has prognostic significance for disease-free and overall survival of patients with HCC.
HEPATOCELLULAR CARCINOMA (HCC) is one of the most common cancers worldwide, especially in Southeast Asia and Africa.1 The annual incidence rate of HCC in Japan is approximately 30 per 100,000 population, and its mortality is ranked third as a cancer death.1 Cirrhosis and chronic hepatitis caused by hepatitis B or C viral infection have been discussed in association with the development of HCC.2–6 Surgical resection is the main modality of treatment for HCC, but the prognosis remains poor even in curatively resected cases: 5-year survival rate is 25% to 50% after surgery, mainly owing to the high recurrence rate.7–11 Several histologic factors have been reported to be prognosticators for HCC. Among them, tumor size and vascular invasion, either portal or hepatic, were the main factors for tumor recurrence7–15; these two factors, together with the multiplicity of the tumor, were included in the tumor-node-metastasis (TNM) classification for HCC.16 However, prognoses of patients with solitary and small-sized tumors without microscopic vascular invasion were still unfavorable.17 Therefore, it is important to identify other prognostic factors responsible for the recurrence of HCC.
Recently, we identified the gene-encoding valosin-containing protein (VCP; also known as p97) as being associated with metastasis of the murine osteosarcoma cell line by using the mRNA subtraction technique.18 VCP, a member of the ATPases associated with various cellular activities (AAA) superfamily, is involved in the ubiquitin-dependent proteasome degradation pathway of inhibitor In this study, expression level of VCP in HCC was examined by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical analysis, and its correlation with recurrence and survival of patients with HCC was evaluated.
Patients One hundred seventy patients who underwent curative resection for primary HCC at the Gastroenterological Surgery Division of Osaka University Hospital in Osaka, Japan, during the period from July 1987 to February 2000 were selected for this study. There were 139 male and 31 female patients, with ages ranging from 31 to 81 years (median, 61 years). Thirty-seven patients were positive for hepatitis B virus surface antigen, whereas 99 patients were positive for hepatitis C virus antibody. Preoperative diagnostic imagining examinations, including ultrasonography, computed tomography scan, and angiography, were performed in all patients. Liver function was assessed by combined findings of Pugh-Child’s classification, liver biochemistry including serum alpha-fetoprotein (AFP) level, and indocyanine green retention test. Preoperative transarterial embolization was performed in 62 patients (36%). According to the number of resected anatomic segments,21 surgical procedures used were classified as follows: limited resection (79 patients), subsegmentectomy (41 patients), segmentectomy (29 patients), lobectomy (16 patients), and extended lobectomy (five patients).
Surgically resected specimens were fixed in 10% formalin and routinely processed for paraffin embedding. Histologic sections cut at 4-µm thickness were stained with hematoxylin and eosin and reviewed by one of the authors (Y.H.) to determine the following categories: differentiation of tumor cells based on the criteria proposed by Edmondson and Steiner (I, well-differentiated; II, moderately differentiated; III, poorly differentiated; IV, undifferentiated),2 pattern of growth (expansive or infiltrative), formation of fibrous capsule around the tumor, portal vein invasion, tumor multiplicity, and positivity for the surgical margin. When tumor cells were present within 5 mm from the edge, surgical margin was defined as positive. Degree of inflammation and fibrosis in noncancerous hepatic tissues were shown as the histologic activity index (HAI) score.22 Regarding inflammatory grade, there were 13 patients with no activity (score of 0), 60 with minimal activity (score of 1 to 3), 84 with moderate activity (score of 4 to 7), and 13 with severe activity (score of After surgery, measurement of serum AFP level and ultrasonography and computed tomography scan were performed at 1- and 3-month intervals, respectively. When tumor recurrence was suspected, angiography was performed. The patients were observed until March 31, 2002; the follow-up periods for survivors ranged from 5 to 136 months (median, 43 months) after surgery.
Immunohistochemical Analysis
Quantitative RT-PCR Analysis of VCP
Statistics
VCP Expression in HCC Eight (4.7%) of 170 sections that did not show endothelial staining by immunohistochemistry were regarded as having poor antigen preservation and were excluded from further analysis. The remaining 162 cases showing endothelial staining were evaluated for VCP expression. Forty-two cases showed a constant level 1 staining in the cytoplasm of HCC in every area of the tumor, whereas 15 cases showed level 2 staining at the peripheral zone but level 1 in the larger central area of the tumor. In total, 57 cases (35.2%) were regarded as having level 1 VCP expression. The remaining 105 cases (64.8%) showed level 2 staining throughout the tumors (Fig 1  ).
Quantitative RT-PCR analysis was performed in four HCCs with level 1 and seven with level 2 expression. Relative ratio of VCP/beta-actin expression in cases with level 1 and 2 expression was 3.1 ± 1.3 and 9.0 ± 4.0 (mean ± SD), respectively (P < .05; Fig 2 ). All but one of level 1 cases showed the lower VCP expression than level 2 cases at the RT-PCR analysis.
Uni- and Multivariate Analyses for Prognostic Factors in HCC Significant difference was observed in the presence of portal vein invasion between HCC with VCP level 1 (40.0%) and level 2 (61.9%) expression (P < .01; Table 1 ). There was no significant relation between VCP level and other clinicopathologic factors.
Five-year disease-free and overall survival rates of the 162 patients with HCC were 26.7% and 64.9%, respectively. Tumor recurrence during the course was found in remnant liver in 109 patients, lymph node in four, bone in two, and other organs in three. Prognostic significance of VCP expression was analyzed for disease-free and overall survival. Patients with level 1 HCC had better 5-year survival rates than those with level 2 HCC (disease-free survival rate, 50.5% v 15.3%; P < .0001; overall survival rate, 79.2% v 57.0%; P < .05; Fig 3 ; Table 2). Twenty-eight (49.1%) of 57 level 1 patients developed recurrent HCC at 32.0 ± 4.2 (mean ± SD) months after the surgery, whereas 90 (85.7%) of 105 level 2 patients developed recurrent HCC at 19.1 ± 2.3 months (P < .01).
Serum AFP level, tumor multiplicity, presence of portal vein invasion, degree of fibrosis in the noncancerous liver tissue (HAI index of 0 v 1 to 4 and 0 to 1 v 3 to 4), and pathologic TNM (pTNM) stage were significant factors for disease-free and overall survival (Table 2  ). Inflammatory status in the noncancerous liver tissues (two categorizations, HAI index of 0 to 3 v > 3 and 0 to 7 v > 7) significantly affected disease-free survival but did not affect overall survival. Multivariate analysis was performed with factors proven to be significant in the univariate analysis, revealing VCP expression level, tumor multiplicity, and degree of fibrosis in the noncancerous liver tissue (different categorizations, HAI index of 0 v 1 to 4 for disease-free; 0 to 1 v 3 to 4 for overall) to be independent prognostic factors for disease-free and overall survival.
Prognostic Significance of VCP Expression in pTNM Classification
To establish appropriate therapeutic modalities for HCC, precise estimation for tumor recurrence is essential. Although conventional TNM staging, which mainly consists of tumor size, vascular invasion, and tumor multiplicity, is a significant prognosticator for disease-free and overall survival of patients with HCC,8,10,11 increased recurrence rates among patients even at low stage (I and II) has been reported.8,10,11 To reinforce the accuracy of prognostication of the TNM staging system, several biologic factors, such as cellular proliferation indices, DNA ploidy pattern in tumor, and aberrant gene expression,27 have been proposed as candidates for prognosticators. However, the prognostic significance of these factors is still controversial among studies.27 This study was conducted to clarify whether VCP expression level could be a new prognostic factor for HCC. Patient characteristics (sex, age, viral association, and 5-year survival rates) in this series were similar to those of previous reports from Japan,8,11 other Asian,9,10,12 and Western countries.7 Uni- and multivariate analyses in this study showed the prognostic significance of tumor multiplicity, serum AFP index, presence of portal vein invasion, pTNM stage, and degree of fibrosis in the noncancerous lesion, as reported previously.8–12,28 These findings indicate that the results obtained from these cases are applicable to HCC in other countries. In this study, VCP expression level was examined in 11 cases by combined quantitative RT-PCR and immunohistochemical analyses and in 151 cases by immunohistochemistry alone. In the 11 cases, there was a clear correlation in VCP expression between the protein (immunohistochemistry) and mRNA (RT-PCR) level, which indicates the reliability of immunohistochemistry for evaluation of VCP expression.
Among the clinicopathologic factors examined, a significant correlation was observed between increased VCP expression and the presence of portal vein invasion of the tumor cells, which was in agreement with our previous study, in which we found that VCP overexpression is correlated with increased metastatic potential of tumor cells in the experimental metastasis model.18 For a successful tumor formation in the portal vein, antiapoptotic pathway including NF Uni- and multivariate analyses revealed the VCP expression level to be an independent prognosticator for HCC recurrence and patient survival. In addition, VCP level proved to be a prognosticator for recurrence of the disease in patients at both the early (I and II) and advanced (III and IV) stages of pTNM classification: the 5-year disease-free survival rate in patients with VCP level 1 and 2 was 64.5% and 21.5% at low stage, respectively, and 22.0% and 0% at high stage, respectively. Combination of VCP level and pTNM classification is a useful tool for prediction of prognosis for patients with HCC. Recently, improvement of prognosis of HCC patients by adjuvant therapies, such as chemotherapy,31,32 immunotherapy,33,34 and combined chemo- and immunotherapy35 has been reported. The grouping system described above could be a valuable guide for choosing various modalities of adjuvant therapy. In patients with early-stage VCP level 1 HCC, favorable outcome could be expected without adjuvant therapy, whereas patients with level 2 and/or advanced-stage HCC should receive intensive treatment with adjuvant therapy. In conclusion, VCP expression as determined by immunohistochemistry could be used as a new prognosticator for HCC. This study shows that stratification of HCC patients based on the stage of disease, tumor multiplicity, and VCP expression level is useful for prediction of prognosis for HCC patients. This system might open a new way to explore effective modalities of treatment for HCC.
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Copyright © 2003 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
(I
8). The degree of fibrosis was categorized as stage 0 (no fibrosis) in 14 patients, stage 1 (mild fibrosis) in 53, stage 3 (severe fibrosis) in 55, and stage 4 (cirrhosis) in 48. 
2 test and Fisher’s exact probability test. Overall and disease-free survival rates were calculated by using the Kaplan-Meier method,
