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First Published Online November 7, 2008
The Oncologist, Vol. 13, No. 11, 1155-1165, November 2008; doi:10.1634/theoncologist.2008-0081
© 2008 AlphaMed Press

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Hepatobiliary

Osteopontin Combined with CD44, a Novel Prognostic Biomarker for Patients with Hepatocellular Carcinoma Undergoing Curative Resection

Guo-Huan Yang, Jia Fan, Yang Xu, Shuang-Jian Qiu, Xin-Rong Yang, Guo-Ming Shi, Bing Wu, Zhi Dai, Yin-Kun Liu, Zhao-You Tang, Jian Zhou

Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, People's Republic of China

Key Words. Osteopontin • CD44 • Tissue microarray • Hepatocellular carcinoma • Prognosis

Correspondence: Jian Zhou, M.D., Ph.D., Liver Cancer Institute, Zhongshan Hospital, Fudan University, 136 Yi Xue Yuan Road, Shanghai 200032, P. R. China. Telephone: 86-21-64037181; Fax: 86-21-64037181; e-mail: zhou.jian{at}zs-hospital.sh.cn

Received May 5, 2008; accepted for publication October 15, 2008; first published online in THE ONCOLOGIST Express on November 7, 2008.

Disclosure: The content of this article has been reviewed by independent peer reviewers to ensure that it is balanced, objective, and free from commercial bias. No financial relationships relevant to the content of this article have been disclosed by the authors, planners, independent peer reviewers, or staff managers.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Author Contributions
 Acknowledgments
 References
 
Background. Osteopontin (OPN) plays important roles in tumor progression and metastasis through binding to CD44 and integrin. The goal of this study was to elucidate the prognostic significance of OPN and CD44 in hepatocellular carcinoma patients.

Methods. Tumor tissue microarray was used to detect the expression levels of OPN and CD44 in 302 hepatocellular carcinoma patients undergoing curative resection between 1997 and 2000 at our institute. Clinicopathologic data for these patients were investigated. The prognostic effects of OPN and CD44 were evaluated using the Kaplan–Meier method and compared using the log-rank test. The Spearman rank test and Fisher's exact test were applied to demonstrate correlations.

Results. Both OPN and CD44 were independent predictors for overall survival and disease-free survival. When OPN and CD44 were taken into consideration together, the predictive range was extended and the sensitivity was improved, especially for those patients with normal serum {alpha}-fetoprotein levels. The 8-year overall survival and disease-free survival rates in OPN+ and/or CD44+ patients were 28.2% and 25.6%, respectively, which were significantly lower than those of OPN–CD44– patients (52.1% and 51.6%, respectively).

Conclusions. OPN combined with CD44 is a promising independent predictor of tumor recurrence and survival in hepatocellular carcinoma patients.


   INTRODUCTION
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Author Contributions
 Acknowledgments
 References
 
Hepatocellular carcinoma (HCC) is currently the sixth most common solid malignancy worldwide and the third leading cause of cancer-related death [1]. Hepatic resection is a potentially curative treatment for HCC, and competes as the first option from an intention-to-treat perspective. Unfortunately, the prognosis remains unsatisfactory mainly as a result of frequent tumor recurrence and metastasis after curative resection. Consequently, it is important to identify the factors that predispose patients to tumor recurrence and death. Discovering sensitive and special prognostic factors may present opportunities for reducing the severity of this disease through early and new therapeutic interventions.

Osteopontin (OPN) and CD44 have been identified as markers for tumor progression [2–8]. Previous studies in our institute have succeeded in identifying OPN as a lead gene and an independent prognostic factor that is overexpressed in metastatic HCC [9, 10]. A growing body of evidence is increasingly lending support to the idea that human cancer can be considered as a stem cell disease [11–13]. CD44 was identified as a tumor stem cell marker in breast cancer [14], prostate cancer [15], colon cancer [16], head and neck cancer [17], and pancreatic cancer [18]. In our previous research, we found OPN and CD44 to be highly expressed in HCC side population (SP) cells that had been confirmed as cancer stem-like cells (submitted data). Recently, clinical and experimental studies suggested that coexpression of OPN and CD44 may stimulate tumor progression and metastasis [19]. In this study, we evaluated the prognostic significance of OPN and CD44 expression in 302 HCC patients following curative resection.


   MATERIALS AND METHODS
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Author Contributions
 Acknowledgments
 References
 
Patients
Three hundred two HCC patients undergoing curative resection between 1997 and 2000 at the Liver Cancer Institute of Fudan University were included in this study. The inclusion criteria of all the patients were: (a) a distinctive HCC diagnosis based on World Health Organization criteria [20, 21], (b) no prior anticancer treatment, (c) a curative resection, (d) suitable formalin-fixed, paraffin-embedded tissues, and (e) complete clinicopathological and follow-up data. Curative resection was defined as: (a) the complete resection of all tumor nodules and the cut surface being free from cancer on histological examination; (b) no cancerous thrombus found in the portal vein (main trunk or two major branches), hepatic veins, or bile duct; (c) no extrahepatic metastasis.

Tumor differentiation was defined according to the Edmondson grading system [22]. Tumor staging was defined according to the sixth edition of the tumor–node–metastasis (TNM) classification of the International Union against Cancer. The clinicopathological characteristics of 302 HCC patients are summarized in Table 1. Ethical approval for human subjects was obtained from the research ethics committee of Zhongshan Hospital, and informed consent was obtained from each patient.


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  		Table 1. The clinicopathological characteristics of 302 cases of HCC

 
Complete follow-up data for these patients (48 women, 254 men), aged 26–75 years (mean, 52), were available. Most of the patients (300) had Child–Pugh class A disease, only two patients had Child–Pugh class B disease. Patients were followed until March 15, 2007. The mean follow-up was 64.5 months (range, 4–121 months). All patients were prospectively monitored using serum {alpha}-fetoprotein (AFP), abdominal ultrasonography, and chest x-ray every 1–6 months after the surgical procedure. Computed tomography (CT) and/or magnetic resonance imaging (MRI) were used to verify tumor recurrence in suspected cases. A diagnosis of recurrence was based on typical imaging appearance on the CT/MRI scan and/or an elevated AFP level. Most deceased patients died from recurrence or metastasis, or associated complications of liver cirrhosis. Overall survival (OS) was defined as the interval between surgery and death or the last observation. The data were censored at the last follow-up for living patients. Disease-free survival (DFS) was measured from the date of resection until the detection of recurrent tumor or the last follow-up assessment. In the DFS analysis, the data were censored for patients without tumor recurrence.

Tissue Microarray Construction
For tissue microarray (TMA) construction, a hematoxylin and eosin (H&E) stained slide was made from each block and was reviewed by two experienced pathologists. Representative tumor regions were morphologically identified and marked on the H&E stained slides. Tissue cylinders with a diameter of 0.6 mm were punched from the marked areas of each block and incorporated into a recipient paraffin block using a precision instrument (Beecher Instruments, Silver Spring, MD), as described previously [23]. To overcome the problem of tissue microheterogeneity and to increase the number of evaluable cases, each donor tissue block was punched two times for the construction of the recipient block. Three different TMA blocks were constructed. In addition, we built a TMA containing 100 cases of peritumoral livers that were randomly chosen from 302 cases. Each TMA contained 200 or 204 cylinders. Consecutive sections of 4 µm in thickness were taken on 3-aminopropyltriethoxysilane-coated slides (Shanghai Biochip Co., Ltd., Shanghai, China). To validate the concordance between TMAs and whole tumor sections, we further detected OPN and CD44 expression for 50 cases randomly chosen from the 302 patients in comparison with whole tumor sections.

Immunohistochemistry
The immunohistochemical examination of OPN and CD44 was performed with monoclonal anti-mouse OPN antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and CD44s antibody (Sigma Chemical Co., St. Louis), respectively. Immunohistochemistry was performed using a two-step immunoperoxidase technique [24, 25]. Briefly, after microwave antigen retrieval, tissues were incubated with primary antibodies for 60 minutes at room temperature. Following half an hour of incubation with secondary antibody, the sections were developed in diaminobenzidine solution under microscopic observation and counterstained with hematoxylin. Negative control slides with the primary antibodies omitted were included in all assays.

Immunohistochemical staining was assessed by three independent investigators without knowledge of the patient characteristics. Discrepancies were resolved by consensus. Scores were assigned as a percentage of positive staining within each cylinder. The mean percentage value of the two cores was considered representative of one tumor. OPN was considered positive if >5% of tumor cells showed moderate or intense staining [26]. CD44 expression was considered positive if moderate or intense staining was present in ≥10% of the tumor cells [25].

Statistical Analysis
The statistical analysis was performed using the statistical package SPSS (version 15.0; SPSS Inc., Chicago, IL). DFS and OS were analyzed using the Kaplan–Meier method and compared using the log-rank test. The Spearman rank test and Fisher's exact test were applied to demonstrate clinicopathological correlations. Univariate and multivariate risk ratios were computed, together with 95% confidence intervals. Differences associated with p-values below 0.05 were considered significant.


   RESULTS
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Author Contributions
 Acknowledgments
 References
 
TMA Expression Levels of OPN and CD44 Are Concordant with Conventional Sections
Staining for OPN in HCC showed cytoplasmic immunoreactivity, especially in areas with a high density of vasculature (Fig. 1A). However, OPN+ cancer cells were not always observed in regions of capsular infiltration. Expression of CD44 in TMAs was present on the cell membrane of HCC (Fig. 1B), but showed a variation in intensity from one tumor to another. As for OPN staining in peritumoral liver tissues, most cases were negative or only showed a few scattered positive cells with faint staining in the whole cylinder, and there was no case of moderate or strong staining >5%. There were only seven cases that showed moderate CD44 staining in peritumoral liver tissues. To validate the concordance between TMAs and whole tumor sections, we further detected OPN and CD44 expression for 50 cases randomly chosen from the 302 patients in comparison with whole tumor sections. We found that the OPN and CD44 expression levels in the whole tumor sections were 98% (49 of 50) in accordance with the results in the TMAs. There was only one case with no concordance, in which strong staining of CD44 (>30% positive cells) was seen in the TMA but weak staining (~5% positive cells) was seen in the conventional section. This case was treated as CD44– according to the results of the conventional section.


Figure 1
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  		Figure 1. Typical expression images of OPN (A) and CD44 (B) immunohistochemistry in TMAs. The expression style of OPN and CD44 was a focal or diffuse pattern in the cytoplasm.

Abbreviations: OPN, osteopontin; TMA, tissue microarray.

 
Clinicopathologic Correlation of OPN and CD44 Expression in HCC
OPN and CD44 expression was positive in 73 (24.2%) and 91 (30.1%) cases, respectively, and 133 (44.0%) cases were OPN+ and/or CD44+, of which 31 (10.3%) cases were OPN+ and CD44+ simultaneously (Table 1).

To elucidate the biologic significance, we correlated the OPN and CD44 expression levels with the clinicopathologic features of HCC. As shown in Table 2, CD44 expression in HCC was associated with serum AFP elevation (p = .000) and tumor number (p = .013), but not with tumor encapsulation, tumor differentiation, or vascular invasion. OPN expression, however, was not correlated with any clinicopathologic feature (p > .05).


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  		Table 2. OPN and CD44 expression status in relation to selected clinicopathologic features in 302 HCC patients

 
Prognostic Significance of OPN and CD44 Expression
The 3-, 5-, and 8-year OS and DFS rates were 71.5% and 56.6%, 41.6% and 57.1%, and 47.0% and 40.2%, respectively, for the entire study population.

OPN was an independent prognostic factor for OS and DFS (p = .044 and p = .030) (Table 3). In the OPN– group, the OS and DFS rates were significantly higher than in the OPN+ group (p = .042 and p = .027) (Fig. 2).


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  		Table 3. Univariate analyses of factors associated with survival and recurrence

 


Figure 2
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  		Figure 2. Prognostic significance assessed using Kaplan–Meier survival estimates and log-rank tests. Comparisons of OS and DFS by OPN (A, B) and CD44 (C, D).

Abbreviations: DFS, disease-free survival; OPN, osteopontin; OS, overall survival.

 
CD44 was also prognostic for OS (p = .000) and DFS (p = .000) (Table 3). The CD44– group had significantly longer OS and DFS times than the CD44+ group. The expression of CD44 in HCC was associated with serum AFP elevation (p = .000) (Table 2). When further stratification was conducted according to serum AFP level, we found that CD44+ patients had shorter OS and DFS times than CD44– patients in both the normal and positive serum AFP groups (p = .000 and p = .000) (supplemental online Fig. S1).

When OPN and CD44 expression were taken into consideration together, the OS and DFS rates were significantly higher in the OPN–CD44– group than in the CD44+OPN–, CD44–/OPN+, and CD44+/OPN+ groups (p < .05) (supplemental online Fig. S2); there was no significant difference among the latter three groups (p > .05) (supplemental online Fig. S2). The 8-year OS and DFS rates in OPN+ and/or CD44+ patients were 28.2% and 25.2%, respectively, which were significantly lower than those of OPN–CD44– patients (52.1% and 51.6%, respectively; p = .000). When stratified by AFP level, we found that OPN+ and/or CD44+ patients had lower OS and DFS rates than OPN–CD44– patients in both the normal and positive AFP groups (p = .000) (Fig. 3).


Figure 3
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  		Figure 3. Kaplan–Meier analysis of OS and DFS for combined OPN and CD44 (A, B) and stratified by AFP level (C–F).

Abbreviations: AFP, {alpha}-fetoprotein; DFS, disease-free survival; OPN, osteopontin; OS, overall survival.

 
Univariate analysis showed that tumor size, TNM stage, vascular invasion, and tumor number were unfavorable predictors for OS and DFS, while age, gender, hepatitis B virus (HBV) and hepatitis C virus (HCV) infection background, and Child–Pugh class had no prognostic significance for OS and DFS (Table 3). Multivariate analyses of factors demonstrated that the coindex (CD44/OPN) was an independent prognostic factor for both OS and DFS (p = .000) (Table 4). Tumor differentiation, tumor number, and vascular invasion were independent prognostic factors for OS. Meanwhile, vascular invasion was an independent prognostic factor for DFS (Table 4).


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  		Table 4. Multivariate analysis of factors associated with OS and DFS (coindex)

 
Sensitivity and Specificity of OPN and CD44 in Predicting Tumor Recurrence
As shown in supplemental online Table S1, the predictive sensitivity and specificity of OPN for tumor recurrence were 27.8% and 80.6%, respectively, and those for CD44 were 40.5% and 83.7%, respectively. Furthermore, when OPN and CD44 were taken into consideration together to predict tumor recurrence, the sensitivity and specificity were 54.9% and 70.5%, respectively. In the normal serum AFP group, those parameters were 68.7% and 61.9%, respectively.


   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Author Contributions
 Acknowledgments
 References
 
Chronic infection with HBV is the primary cause of HCC in China, whereas HCV plays a more prominent role in western countries and Japan [27]. Despite improvements in surveillance and clinical treatment strategies, the prognosis of HCC still remains dismal [28, 29]. It is critical to classify those patients with a high probability of tumor recurrence and metastasis well in advance to initiate timely intervention. Current clinicopathologic factors, such as AFP, TNM stage, and tumor size, cannot accurately predict the outcome of all HCC patients [30]. Furthermore, it is very difficult to predict prognosis in AFP-normal and early-stage HCC patients [30, 31]. Studies are needed to explore and identify novel strategies for the prediction of prognosis and therapeutics of HCC.

OPN, a secreted noncollagenous and sialic acid–rich phosphoprotein, was initially discovered as an inducible marker of transformation of epithelial cells, and was later shown to be frequently overexpressed in many human cancers [2], including HCC [3]. Our previous work identified OPN as a lead gene that is overexpressed in metastatic HCC and is able to act as both a diagnostic marker and a potential therapeutic target for metastatic HCC [9]. The level of plasma OPN acted as an independent prognostic factor for both OS and DFS in HCC patients following liver resection [10]. With details pertaining to the exact mechanisms being elucidated, OPN functions through its interaction with the CD44 family of cell surface receptors and integrin. OPN contains a COOH-terminal fragment, which binds directly to CD44. The interaction of OPN, CD44, and β1-integrin was demonstrated to be associated with cell spreading, chemotactic behavior, and metastasis in an experimental model [32]. Clinical and experimental studies suggest that coexpression of OPN and CD44 may stimulate tumor progression and metastasis. Their interaction plays a crucial role in lymph node metastasis of gastric carcinoma [19]. Despite growing evidence for the role of both OPN and CD44 in tumorigenesis/metastasis, how they cooperate to promote tumor progression remains poorly understood. Cancer stem cells for breast, prostate, colon [16], head and neck, and pancreatic [18] cancer have been described, lending support to the 40-year-old idea that cancers may originate from a common progenitor. Thus, evidence is accumulating in support of the view that cancer may actually be a single disease [11, 13, 16]. Several tumors, including HCC, were shown to contain cancer stem cells using the cell surface marker CD44 [33, 34]. Studies in our laboratory also demonstrated that OPN and CD44 were highly expressed in SP cells from HCC cell lines and human HCC specimens, which were confirmed as cancer stem-like cells.

In this study, we found that both OPN and CD44 were independent predictors of OS and DFS in HCC patients. The rates of OPN and CD44 expression were 24.2% and 30.1%, respectively, and 10.3% of patients expressed both OPN and CD44. Interestingly, we found that the predictive range was extended and the sensitivity was improved when both OPN and CD44 were taken into consideration together. More than 40% of patients in this study expressed OPN or CD44, and were more prone to recurrence of HCC following curative resection. On multivariate analysis, the coindex CD44/OPN was found to be an independent prognostic factor for both OS and DFS. The OS and DFS rates in OPN–CD44– patients were significantly higher than those in OPN+ and/or CD44+ patients.

Furthermore, OPN+ and/or CD44+ patients had unfavorable OS and DFS times in both the normal and positive serum AFP groups, when compared with OPN–CD44– patients. Serum AFP remains the most widely used tumor marker in the diagnosis and management of HCC [35]. It has a sensitivity of 39%–65%, a specificity of 76%–94%, and a positive predictive value of 9%–50% [36]. Nevertheless, there is no ideal factor to demonstrate its prognostic value in the 30%–40% of HCC patients with normal serum AFP levels. In our study, CD44 tended to be highly expressed in patients with negative AFP. OPN positivity and/or CD44 positivity, endowed with a sensitivity of 68.7% and a specificity of 61.9%, showed significant value in predicting tumor recurrence for the HCC patients with normal serum AFP levels.

It has been suggested that HCC has two patterns of recurrence—multicentric occurrence and intrahepatic metastasis. Multicentric HCCs also show synchronous and metachronous occurrence [37, 38], and the latter is thought to be clinically more important. In terms of tumor differentiation, an Edmondson grade I or II tumor has a low incidence of intrahepatic metastasis before curative resection. Furthermore, long-term recurrence (>2 years) has different meanings than short-term recurrence (≤2 years); the former is always thought to be associated with multicentric occurrence. In this study, tumor recurrence was found in 40.5% (70 of 173) of patients 2 years postoperatively. Fifty-one patients with Edmondson grade I or II tumors recurred after 2 years (44.0%, 51 of 116); to our surprise, OPN and CD44 were positive in 80.4% (41 of 51) of these patients. That is to say, OPN plus CD44 may be very sensitive in predicting metachronous occurrence of HCC after curative resection.

In conclusion, OPN combined with CD44 might serve as an unfavorable prognostic factor for HCC patients undergoing curative tumor resection. Thus, undertaking adjuvant treatment early in OPN+ and/or CD44+ patients after operation may reduce tumor recurrence and prolong survival. The underlying mechanism, which might involve cancer stem cells, still needs to be further investigated.


   AUTHOR CONTRIBUTIONS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Author Contributions
Acknowledgments
 References
 
Conception/design: Yang Xu, Jian Zhou

Financial support: Yang Xu, Jian Zhou

Administrative support: Jia Fan, Yin-Kun Liu, Zhao-You Tang

Provision of study materials: Yang Xu, Jia Fan, Shuang-Jian Qiu, Bing Wu, Zhi Dai, Yin-Kun Liu, Zhao-You Tang, Jian Zhou

Collection/assembly of data: Guo-Huan Yang, Xin-Rong Yang, Guo-Ming Shi

Data analysis: Guo-Huan Yang, Xin-Rong Yang, Guo-Ming Shi

Manuscript writing: Guo-Huan Yang, Yang Xu, Jian Zhou

Final approval of manuscript: Jian Zhou


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Author Contributions
 Acknowledgments
References
 
This study was supported by grants from the National Natural Science Foundation of China (No. 30571801, 30500594), Shanghai Science and Technology Development Funds (No. 054119530, 06QA14012), and National Hi-Tech Research and Development Program of China (No. 2007AA02Z479). We thank Shanghai Biochip Co., Ltd. for help in construction of the tissue microarrays.

Guo-Huan Yang, Jia Fan, and Yang Xu contributed equally to this work


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Author Contributions
 Acknowledgments
 References
 

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THE ONCOLOGIST STEM CELLS CME ALPHAMED PRESS JOURNALS