Mol Biol Rep (2012) 39:351–357 DOI 10.1007/s11033-011-0745-y
Glypican-3, a novel prognostic marker of hepatocellular cancer, is related with postoperative metastasis and recurrence in hepatocellular cancer patients Su Ning • Chen Bin • Huang Na • Shen Peng Ding Yi • Ye Xiang-hua • Zeng Fang-yin • Zheng Da-yong • Luo Rong-cheng
•
Received: 10 May 2010 / Accepted: 27 April 2011 / Published online: 8 June 2011 Ó Springer Science+Business Media B.V. 2011
Abstract Metastasis/recurrence has been the most fundamental characteristic of hepatocellular cancer (HCC) and the ultimate cause of most HCC-related deaths. However, there are still a limited number of reliable tumor markers that can be used to predict the possibility of metastasis/ recurrence in an HCC patient after operation. Recently, much evidence has shown that glypican-3 (GPC3) can be a useful tool to identify the early development of HCC, but little research has been done to test its usefulness as a prognostic marker related to post-operative metastasis/ recurrence in HCC patients. In this study, the expression of GPC3 and its relationship with clinicopathological factors were determined by immunohistochemical analysis in 61
primary HCC patients. The potential prognostic value of GPC3 was investigated by comparing the survival time between HCC patients with high and low GPC3 expression. The results demonstrated that GPC3 expression was closely related with metastasis/recurrence in an HCC patient who can receive the operation. The risk of metastasis/recurrence after surgery in an HCC patient with high GPC3 expression was increased to 3.214 as compared to that of an HCC patient with low GPC3 expression. Survival analysis showed that HCC patients with high GPC3 expression had a significantly shorter overall survival time than HCC patients with low GPC3 expression (P = 0.003). Further, multivariate analysis showed that GPC3 expression was a significant, independent prognostic parameter
Su Ning and Chen Bin contributed equally to this work. S. Ning C. Bin H. Na S. Peng Z. Da-yong (&) L. Rong-cheng (&) Department of Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, People’s Republic of China e-mail:
[email protected] L. Rong-cheng e-mail:
[email protected] S. Ning e-mail:
[email protected] C. Bin e-mail:
[email protected] H. Na e-mail:
[email protected] S. Peng e-mail:
[email protected]
Y. Xiang-hua e-mail:
[email protected] Z. Fang-yin Department of Clinical Laboratory, School of Basic Medical Sciences, Southern Medical University, Guangzhou, Guangdong, People’s Republic of China e-mail:
[email protected] S. Ning Department of Oncology, Guangzhou Chest Hospital, Guangzhou, Guangdong, People’s Republic of China C. Bin General Hospital of Guangzhou Military Command of PLA, Guangzhou, Guangdong, People’s Republic of China
D. Yi Y. Xiang-hua Department of Radiation Oncology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, People’s Republic of China e-mail:
[email protected]
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(P = 0.030) for HCC patients. Overall, the study indicates that GPC3 might be a valuable marker closely related with prognosis and post-operative metastasis/recurrence in HCC patients. Keywords Hepatocellular carcinoma Glypican-3 Metastasis Recurrence Prognosis
Background Hepatocellular carcinoma (HCC), a common solid malignancy, is the third most common cause of cancer-related mortality worldwide [1]. However, the prognosis of HCC patients remains poor, with its 5 year survival rate after surgery as low as 25–39% [2]. Surgical resection remains to be the standard choice of treatment for patients in the early stage of HCC. However, even with radical resection, 60–70% of patients develop metastasis and recurrence within 5 years after surgery [3, 4]. Although several clinicopathological features including a poorly differentiated phenotype, large-sized tumor, and portal venous invasion have been shown to contribute to the poor prognosis in HCC patients before operation, there remain a limited number of reliable tumor markers that can be used to predict the possibility of metastasis/recurrence in an HCC patient after operation. Glypican-3 (GPC3) is a member of the glypican family of heparan-sulfate proteoglycans (HSPGs), which is bound to the plasma membrane through a glycosyl phosphatidylinositol (GPI) anchor. GPC3 is highly expressed in HCC, and it has been discovered as a potential serological and histochemical marker specific for the differentiation between the early stage of HCC formation and its precancerous state [5]. For example, a dramatic elevation of GPC3 mRNA and protein levels has been reported in large amounts of HCC, whereas its expression was less frequent in preneoplastic or entirely absent in non-neoplastic liver [6–9]. Studies have shown that GPC3 can stimulate the growth of HCC cells by up-regulating autocrine/paracrine canonical Wnt signaling, and regulate migration, adhesion in tumor cells. We have hypothesized that GPC3 overexpression might be closely related with prognosis and post-operative metastasis/recurrence in HCC patients. However, until now, the clinical significance of GPC3 over-expression in HCC remains to be vague, which includes the relationship between GPC3 over-expression and post-operative metastasis/recurrence. In this study, the relationship of GPC3 expression with its corresponding clinicopathological features, especially that between GPC3 expression and the prognosis of HCC patients, was examined using immunohistochemical analysis.
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Materials and methods Patient tissue samples A total of 61 patients with primary HCC who underwent hepatectomy at Nanfang Hospital (Guangzhou City, Guangdong Province, China) between 1996 and 2003 were enrolled in this study. They were not pretreated with radiotherapy or chemotherapy prior to surgery. They were followed up for over 5 years until June 2008, and their complete clinical data were collected. There were 55 men and six women with a mean age of 48.0 ± 11.5 years (range: 28–71 years). Of these patients, 53 (80.3%) were positive for hepatitis B surface antigen (HBsAg).Tumor size was based on the largest dimension of the tumor specimen. Tumor stage was determined according to the UICC TNM classification system (sixth edition, http://tnmuicc.org). Tumor differentiation was classified using the Edmondson grading system. All patients provided written informed consent before enrollment in this study. And this study was approved by our hospital ethics committee and complied with GCP guidelines and the Declaration of Helsinki and local laws. All primary HCC tissue samples were fixed in 10% formalin, embedded in paraffin, sectioned consecutively at 4 lm, and stained by hematoxylin and eosin. In a doubleblinded manner, the histological types were assigned independently by three pathologists according to the criteria of the World Health Organization classification system.
Immunohistochemistry Paraffin-embedded tissue sections were deparaffinized with xylene and rehydrated in a graded series of ethanol solutions. An antigen-retrieval technique was performed by heating the slides in 0.01 mol/l, pH 6.0 sodium citrate buffer in a microwave oven for 13 min at 850 w, which was repeated twice. Then endogenous peroxidase was blocked with 0.3% H2O2 methanol for 15 min at room temperature. Immunostaining was performed using mouse anti-GPC3 (sc-65443, Santa Cruz Biotechnology, CA) with a dilution of 1:300 at 4°C overnight. Serial sections were stained with a horseradish peroxidase enzyme-labeled polymer with anti-mouse/rabbit immunoglobulin according to the instructions of the Chemmate EnVision/Mo&Rb detection kit (GK500705, Gene Tech Company Limited, Shanghai, China). Finally, the visualization signal was developed with 3,3-diaminobenzidine tetrahydrochloride (AR1022, Boster Biological Tech., Ltd., Wuhan, China), and the slides were counterstained in hematoxylin. PBS was used as a negative control.
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Evaluation of staining of GPC3 The total GPC3 immunostaining score was calculated as the sum of percent positivity of the stained tumor cells and the staining intensity. The percent positivity was scored as ‘‘0’’ (\5%), ‘‘1’’ (5–10%), ‘‘2’’ (11–50%), and ‘‘3’’ ([50%). The staining intensity was scored as ‘‘0’’ (no staining), ‘‘1’’ (weakly stained), ‘‘2’’ (moderately stained), and ‘‘3’’ (strongly stained). Both percent positivity of the cells and staining intensity were decided in a double blinded manner. The final GPC3 expression score was calculated with the value of percent positivity score 9 staining intensity score, which ranged from 0 to 9. Then the expression level of GPC3 was defined as ‘‘-’’ 0, ‘‘?’’ 1–2, ‘‘??’’ 3–5, and ‘‘???’’ 6–9 [10, 11]. Statistical analysis All statistical analyses were performed by SPSS13.0 for Windows (SPSS Inc., Chicago). The correlation between GPC3 expression and various clinicopathological parameters in the tissue samples was evaluated with v2 test or Fisher’s exact test (when the number of samples is less than five, it would be Fisher’s exact test, otherwise v2 test). The overall survival was calculated from the date of surgery to the date of death. Survival analyses were carried out according to the Kaplan–Meier method, and the differences were assessed using the log-rank test. Cox proportionalhazards analysis was used for univariate and multivariate analyses to explore the effects of the variables on survival. A P value \0.05 was considered significant.
Results Expression of GPC3 in HCC tissues GPC3 expression level was examined in 61 of the surgical specimens of HCC. The results showed that GPC3 was highly expressed in 32 (52.5%) cases, but no or low GPC3 expression was detected in the remaining 29 cases (47.5%). In most GPC3-positive cases, the immunoreactivity of GPC3 was chiefly localized in the cellular cytoplasm, with a certain amount detected in the cell membrane (Fig. 1). The expression of GPC3 was evaluated in relation to the clinicopathological features and follow-up data. GPC3 expression was closely related with hepatic cirrhosis, histological differentiation, TNM staging, and metastasis/ recurrence (Table 1). HCC patients with hepatic cirrhosis had a higher over-expression rate than those with no hepatic cirrhosis. GPC3 expression was less frequently observed in well or moderately differentiated HCC than in poorly differentiated HCC (Fig. 1). In addition, the rate of
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expression of GPC3 protein in patients with poorly differentiated HCC was higher than that with well or moderately differentiated HCC. The over-expression rates of GPC3 in advanced HCC patients (TNM staging III ? IV) were higher than those in early HCC patients (TNM staging I ? II). Compared with HCC patients with low GPC3 expression, the risk of metastasis/recurrence after surgery in HCC patients with high GPC3 expression was increased to 3.214. No significant correlation was found between GPC3 expression and gender, age, tumor size, serum HBsAg, and serum AFP (Table 1). Correlation between GPC3 expression and patients’ survival Through Kaplan–Meier curve assessment, patients with increased GPC3 expression were observed to have a worse prognosis than those with decreased GPC3 expression (Fig. 2). The median survival time of HCC patients with GPC3 over-expression was 17.0 months, while that of HCC patients with low GPC3 expression was 41.0 months. The difference was statistically significant (P = 0.003, logrank test). Univariate and multivariate analyses to identify the prognostic variables in HCC patients Univariate analysis with stepwise inclusion of variables in the model showed that GPC3 expression, serum AFP, tumor size, metastasis/recurrence, and TNM staging were the significant prognostic factors. Furthermore, the multivariate analysis results showed that GPC3 expression (P = 0.030), serum AFP (P = 0.004), tumor size (P = 0.022), and metastasis/recurrence (P = 0.000) might play a role in predicting the overall survival in HCC patients (Table 2).
Discussion HCC, one of the most aggressive types of cancer, is associated with poor prognosis despite significant advancements in surgical and locoregional therapies over the past two decades. Considerable interest has recently been focused on the mechanism of HCC metastasis/recurrence after operation, specifically the most fundamental characteristics of HCC and the ultimate cause of most HCC mortalities. Thus, screening more potential early prognostic markers and therapeutic targets is very urgent for oncologists. GPC3 is involved in the migration, proliferation, and modulation of cell survival in several tissues [12–15]. Glypicans are released from the cell surface by a lipase called Notum, whose functions may regulate the signaling
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Fig. 1 Expression of GPC3 in HCC tissues. a Negative expression in HCC tissue (IHC 9 400). b Weak expression in HCC tissue (IHC 9 400). c Strong expression in moderately differentiated HCC tissue
(IHC 9 400). d Negative expression in normal live tissue (IHC 9 400). e HCC tissue (H&E 9 400). f Strong expression in poorly differentiated HCC tissue (IHC 9 200)
of Wnts, Hedgehogs, fibroblast growth factors, and bone morphogenetic proteins pathways [16–20]. The expression of the GPC3 protein is found to be stronger in HCC tissues than in normal liver and cirrhotic liver tissues [7, 21]. Furthermore, serum GPC3 levels are also higher in HCC patients than in any other liver disease patients [8, 9]. GPC3 was introduced as a potential marker for HCC by observing significantly high levels of this protein in the serum of HCC patients, whereas nearly undetectable in the serum of healthy donors and patients with benign liver
diseases. Recently, Shirakawa et al. [22] found the potential value of GPC3 as a prognostic molecular biomarker for patients with hepatocellular carcinoma. Kwack et al. [23] also discovered that GPC3 could reduce the adhesion of HCC cells to collagen type I and fibronectin, enhancing cellular migration and invasiveness. In this study, GPC3 expression in HCC was examined, and the clinical prognostic value of GPC3 was explored using the complete long-term follow-up data of 61 HCC patients and corresponding clinical samples. The tabular
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Table 1 Relationship between glypican-3 expression and clinicopathologic features of HCC patients Features
n
GPC3 expression High (%)
Low(%)
61
32 (52.5%)
29 (47.5%)
Male
55
29 (52.7%)
26 (47.3%)
Female
6
3 (50.0%)
3 (50.0%)
\50
36
20 (55.6%)
16 (44.4%)
C50
25
12 (48.0%)
13 (52.0%)
\5 cm
24
12 (50.0%)
12 (50.0%)
C5 cm
37
20 (54.1%)
17 (45.9%)
Positive
53
26 (49.1%)
27 (50.9%)
Negative
8
6 (75.0%)
2 (25.0%)
All cases Gender
P value
1.000
Age
0.561
Tumor size
0.757
Serum HBsAg
0.260
Serum AFP
0.256
\25 ng/ml
29
13 (44.8%)
16 (55.2%)
C25 ng/ml
32
19 (59.4%)
13 (40.6%)
Yes
46
28 (60.9%)
18 (39.1%)
No
15
4 (26.7%)
11 (72.3%)
Hepatic cirrhosis
0.021
Histological differentiation
0.036
Well/moderately
45
20 (44.4%)
25 (55.6%)
Poorly
16
12 (75.0%)
4 (25.0%)
I ? II
45
20 (44.4%)
25 (55.6%)
III ? IV
16
12 (75.0%)
4 (25.0%)
Yes
38
24 (63.2%)
14 (36.8%)
No
23
8 (34.8%)
15 (65.2%)
TNM staging
0.036
Metastasis/recurrence
0.031
analysis showed that GPC3 expression was correlated not only with metastasis/recurrence in HCC patients after operation but also with the state of hepatic cirrhosis, histological differentiation, and TNM. The Kaplan–Meier survival analysis revealed that a high level GPC3 expression was significantly linked to the poor prognosis of HCC patients after surgical resection. By univariate analysis of the Cox proportional-hazard model, GPC3 expression, serum AFP, tumor size, metastasis/recurrence, and TNM staging were found to be associated with an increased risk of death by HCC. This finding is in agreement with previous reports [24]. Thus, GPC3 expression may function as a new and independent predictor of prognosis for HCC patients. A high level GPC3 expression indicates poorer prognosis in patients with HCC. In addition, HCC patients with hepatic cirrhosis were also found to have a higher GPC3 over-expression rate than
Fig. 2 Kaplan–Meier survival analysis of primary HCC patients (n = 61) after surgical resection with high GPC3 expression (n = 32) and low GPC3 expression (n = 29). The survival rate for patients in the GPC3-high group was significantly lower than that for patients in the GPC3-low group (P = 0.003, log rank)
non-hepatic cirrhosis HCC patients. High levels of GPC3 expression were observed in poorly differentiated tumor tissues, consistent with previous reports [6, 22]. Hippo et al. [25] discovered that soluble GPC3 (sGPC3), the NH2-terminal portion of GPC3, seems to be better than AFP in terms of sensitivity in detecting well or moderately differentiated HCC. Thus, the GPC3 expression level was correlated with HBV-related hepatic cirrhosis and the tumor differentiation level. GPC3 might also play a role in hepatocarcinogenesis. According to 2002 AJCC/UICC HCC TNM staging, the over-expression rates of GPC3 in advanced HCC patients (TNM staging III ? IV) were higher than those in early HCC patients (TNM staging I ? II). Compared with HCC patients with GPC3 down-expression, HCC patients with GPC3 over-expression have higher risks of post-operative metastasis/recurrence (OR = 3.214). There are two reasons related to the post-operative metastasis/recurrence manner: intrahepatic metastasis in the residual liver in a metaoperative metastasis/recurrence and multicentric hepatocarcinogenesis based on chronic hepatitis [26]. GPC3 expression has a close relationship not only with HBV-related hepatic cirrhosis, which may induce HCC recurrence, but also with the tumor differentiation level in response to the malignant degree of tumor cells. These results indicate that GPC3 expression may indicate a high risk of metastasis/recurrence of HCC. In summary, this study evaluated the prognostic significance of GPC3 expression at the protein level in clinical tissue of HCC by immunohistochemical analysis. HCC patients with high GPC3 expression were found to have a
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Table 2 Univariate and multivariate analyses of individual parameters for correlations with overall survival rate: Cox proportional hazards model Variables
Univariate HR
P value
CI (95%)
Multivariate
P value
HR
CI (95%)
1.905
1.063–3.415
0.030
GPC3 expression (1 = down, 2 = over)
2.297
1.297–4.067
0.004
Gender (1 = male, 2 = female)
0.358
0.111–1.156
0.086
Age (1 \ 50, 2 C 50) Serum HBsAg (1 = negative, 2 = positive)
0.782 1.550
0.439–1.396 0.612–3.926
0.406 0.355
Serum AFP (1 \ 25 ng/ml, 2 C 25 ng/ml)
2.035
1.148–3.608
0.015
2.508
1.346–4.674
0.004
2.052
1.109–3.798
0.022
Tumor size(1 \ 5 cm, 2 C 5 cm)
1.820
1.008–3.288
0.047
Histological differentiation (1 = poorly, 2 = well/moderately)
0.869
0.461–1.636
0.663
Hepatic cirrhosis (1 = no, 2 = yes)
1.475
0.754–2.884
0.257
Metastasis/Recurrence (1 = no, 2 = yes)
3.231
1.711–6.099
0.000
4.675
2.251–9.708
0.000
TNM staging(1 = I ? II, 2 = III ? IV)
2.039
1.103–3.769
0.023
1.218
0.633–2.343
0.556
HBsAg hepatitis B surface antigen, AFP alpha-fetoprotein, HR hazard rating, CI confidence interval
significantly poorer prognosis. The most valuable finding is that GPC3 expression not only correlated closely with hepatic cirrhosis, histological differentiation, and TNM staging but also with post-operative metastasis/recurrence, which is vital to the successful treatment of liver cancer. Compared with HCC patients with low-expressed GPC3 protein, the risk of post-operative metastasis/recurrence in HCC patients with GPC3 protein high-expression was increased to 3.214. This result suggests that GPC3 may be used as a valuable marker in evaluating the possibility of metastasis/recurrence for a patient receiving operation. Thus, closer monitoring and a more aggressive treatment should be indicated for HCC patients with high GPC3 expression. The effect of hepatocarcinogenesis prevention after surgery in patients with GPC3-positive HCC may be expected through anticancer immunotherapy [27–30]. Meanwhile, the effect of post-operative metastasis/recurrence prevention may be expected in HCC patients with high GPC3 expression. Acknowledgment This work was grant sponsored by National Natural Science Foundation of China; Grant numbers: 30801380. Guangdong Provincal Science Foundation; Grant numbers: 1045100 1002005047, 8451051501001344.
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