Author Affiliations: Department of Hepatobiliary Surgery, Peking University People's Hospital, Beijing 100044, China (Cui X, Li Z, Gao PJ, Gao J and Zhu JY)

Corresponding Author: Ji-Ye Zhu, MD, Department of Hepatobiliary Surgery, Peking University People's Hospital, Beijing 100044, China (Tel: +86-10-88324175; Fax: +86-10-68310585; Email: gandanwk@vip.sina.com)

 

© 2015, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(15)60349-6

Published online March 13, 2015.

 

 

Acknowledgements: We thank the generous help from Drs. Li-Hua Qian and Jing-Qiu Song, two pathologists from pathology department of Peking University People's Hospital.

Contributors: CX, LZ and ZJY proposed the study. CX and LZ performed the research and wrote the first draft. CX, LZ, GPJ and GJ collected the data. CX analyzed and interpreted the data. CX and LZ contributed equally to this article. ZJY is the guarantor.

Funding: This study was supported by grants from the Special Fund Research of the Ministry of Health (2010.201002015), Specialized Research Fund of the Ministry of Education (20110001110044) and Beijing Key Laboratory Special Fund (Z141107004414042).

Ethical approval: This study was approved by the Ethics Committee of Peking University People's Hospital.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: Glypican-3 (GPC-3) is frequently overexpressed in hepatocellular carcinoma (HCC). Recent studies have shown that GPC-3 is a highly efficient diagnostic biomarker of HCC and an indicator of poor prognosis in HCC patients who have undergone hepatectomy. However, its prognostic value in patients with HBV-associated HCC after liver transplantation (LT) is not clear. The present study is to evaluate the prognostic value of GPC-3 in patients with HBV-associated HCC after LT.

 

METHODS: A cohort of 104 HCC patients with HBV-associated cirrhosis who had undergone LT at our hospital between 2002 and 2011 were enrolled in this study. Samples of HCC were taken from these patients. GPC-3 protein expression was detected in paraffin-embedded specimens using immunohistochemistry. All related clinical data were obtained from the China Liver Transplant Registry. The relationship between GPC-3 expression and clinicopathological parameters was analyzed. Univariate and multivariate Cox-regression analyses were used to identify risk factors for poor prognosis.

 

RESULTS: GPC-3 was expressed in samples from 74 (71.2%) of the 104 patients. GPC-3 was expressed only in HCC cells. Positive staining was correlated with tumor size (P=0.004), encapsulation (P=0.018), pathological stage (P=0.027), portal vein invasion (P=0.043), tumor differentiation (P=0.002) and the Milan criteria (P=0.016). The 5-year survival rate and disease-free survival rate of patients with GPC-3-positive were lower than those (38.2% vs 75.4%, P<0.001; 30.8% vs 69.7%, P=0.001) of patients with GPC-3-negative. Multivariate Cox-regression analysis revealed that GPC-3 was an independent risk factor for 5-year survival rate (P=0.031) and disease-free survival rate (P=0.047), together with tumor differentiation, Milan criteria and pre-operative alpha-fetoprotein.

 

CONCLUSION: GPC-3 is a potential biomarker for poor prognosis after LT in HCC patients with HBV-associated cirrhosis.

 

(Hepatobiliary Pancreat Dis Int 2015;14:157-163)

 

KEY WORDS: liver transplantation; hepatitis B; hepatocellular carcinoma; glypican-3

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the third most common cause of cancer death worldwide.^[1] Although there are many diagnostic and therapeutic options currently available, HCC still has a poor prognosis, with a low 5-year survival rate. Liver transplantation (LT) is the best treatment option for HCC because it can completely remove both tumors and cirrhotic liver. The rate of HCC recurrence after LT varies from 8% to 20%.^{[2, 3]} Clinicopathological features as predictors of poor prognosis in HCC patients include cancer cell differentiation, large tumors, alpha-fetoprotein (AFP), portal vein invasion and intrahepatic metastasis.

 

Glypican-3 (GPC-3) is a member of the glypican family of glycosyl-phosphatidylinositol-anchored cell-surface heparan sulfate proteoglycans.^[4] Its function was first reported when a mutation in its gene was found to be associated with human Simpson-Golabi-Behmel syndrome.^[5] Its role in cancer appears to vary depending on the cellular context and signaling pathways. GPC-3 is documented to be involved in the tumor growth factor, Wnt/β-catenin, Hedgehog, bone morphogenetic protein and fibroblast growth factor signaling pathways.^[6-11] GPC-3 plays different roles in different cancers. GPC-3 acts as an inhibitor of cell proliferation and pro-apoptotic factor in mesothelioma, and breast and ovarian cancer.^[12] However, GPC-3 overexpression is widespread in HCC, where it acts as an oncogene.^[13] GPC-3 is a diagnostic molecular marker of HCC, which can be used to distinguish HCC from human cholangiocellular carcinoma, focal nodular hyperplasia, hepatocellular adenoma and cirrhosis.^[14-16] GPC-3 is a potential prognostic biomarker for HCC patients who have undergone hepatectomy.^{[17, 18]} The value of GPC-3 for prognosis has been studied in small-scale investigations of post-LT patients with HCC originating from diverse causes.^[19] Antibody targeting GPC-3 has been tested in phase II clinical trials.^[14]

 

Most HCC patients in China are HBV-associated. In our organ transplantation center, HCC patients with HBV-associated cirrhosis comprise the largest group of patients undergoing LT. In the present study, we measured tumor expression of GPC-3 in HCC patients with HBV-associated cirrhosis who underwent LT. Hepatic GPC-3 expression was measured using immunohistochemistry, and the data were compared to prognosis, pre-operative AFP, tumor size and number, Child-Pugh scores, Milan criteria, histological differentiation and MELD scores, which are all established predictors of survival time and recurrence rates in HCC patients after LT.^{[20, 21]}

 

 

The samples were from HBV-associated HCC patients who underwent LT at Peking University People's Hospital from 2002 to 2011. HBV infection was confirmed by serum HBsAg, HBcAb, HBeAg and HBV-DNA. Patients with HCV infection, alcoholic liver disease or extrahepatic metastasis confirmed by CT, MRI or PET were excluded. A total of 104 patients with HBV-associated HCC were enrolled. Liver tumor and adjacent tissue specimens were taken from resected livers. Paraffin-embedded blocks were retrieved from the Department of Pathology of our hospital. Tumor stages were determined according to the TNM system of the International Union Against Cancer and American Joint Committee on Cancer.^[22] The histological grade of each tumor was determined based on the Edmondson-Steiner grading system.^[23]

 

Patients enrolled in the study had detailed medical records for age, gender, height, weight, etiology of hepatitis, pre-operative AFP, Child-Pugh scores, MELD scores, Milan criteria status, number and size of tumor nodules, portal vein tumor thrombosis (PVTT), TNM staging and tumor grade (I-IV). All patients received post-operative immunosuppressive therapy (tacrolimus was initiated 3 months after LT). The patients were followed up post-operatively at the outpatient clinic in our center every 3 months and the follow-up covered HBV DNA, serum AFP, liver function and abdominal ultrasonography. CT was done every 6 months in the first year and once a year thereafter. The patients were given antiviral therapy if their HBV DNA exceeded 1.0×10³ copies/mL. Survival time was defined as the last time the patient had been confirmed to be alive. The study endpoint was March 31, 2014. Cumulative survival time was calculated from the day of operation until patient death or the end of the study. Informed consent was obtained from all subjects and the deceased donor livers were obtained through social and legal donation. This study was approved by the Ethics Committee of Peking University People's Hospital.

 

Paraffin-embedded blocks were sectioned at 3-µm thickness. The Polink-1 HRP DAB Detection System one-step polymer detection method was used. The sections were deparaffinized in xylene and rehydrated through ethanol to water. Then 3% H₂O₂ was used to block endogenous peroxidase activity for 5 minutes. For antigen retrieval, the sections were heated with a high pressure cooker in 10 mmol/L citrate buffer (pH 6.0) for 2.5 minutes, washed in PBS, and cooled. And then they were incubated with prediluted primary monoclonal anti-GPC-3 antibody (rabbit anti-human GPC-3, 1:100, Abcam Co., Hong Kong SAR, China). Primary antibody was not added to the negative control slide in this step. Slides were incubated for 12 hours at 4 ��, washed 3 times with PBS, and then incubated for 30 minutes with secondary antibody at working dilutions (rat anti-rabbit IgG, Beijing Xiya Jinqiao Biology Technology Company, Beijing, China) at room temperature. For color development, DAB was added to slides for 2 minutes and then the slides were counterstained with hematoxylin, dehydrated, cleared and mounted.

 

GPC-3 tissue staining, including HCC lesions and adjacent non-cancerous areas, was independently and blindly evaluated and semi-quantified by two senior pathologists. Seventy-four samples were positive for GPC-3 staining. Immunohistochemical staining was classified according to the relative number of GPC-3-positive stained cells as follows:^[18] negative (<10%), positive (≥10%) (Fig. 1). All samples were divided into GPC-3-negative (<10%) and GPC-3-positive (≥10%) groups based on the relative number of immunoreactive cells and they were assessed as focal lesions.

 

The patients' database was built using SPSS v17.0 software (Chicago, IL, USA). The role of GPC-3 expression in the prognosis of patients with primary HBV-associated HCC was evaluated using the correlation of GPC-3 expression to different clinicopathological factors. All continuous variables are presented as means±standard derivation (SD), as evaluated with the Mann-Whitney U test. The Chi-square test was used to compare qualitative variables. Survival curves were plotted using the Kaplan-Meier method and compared with the log-rank test to determine statistical significance. Multivariate Cox-regression analysis was done to evaluate the independent risk factors associated with prognosis, such as GPC-3 staining, TNM staging, histological differentiation, pre-operative AFP, Milan criteria, tumor size and portal vein invasion. A P<0.05 was considered statistically significant.

 

 

The 104 patients received full-size grafts from deceased donors. After LT, no patient was lost to follow-up. Patient and tumor sample characteristics are shown in Table 1. The median follow-up time was 31.1 months. The 5-year overall survival rate and post-operative tumor recurrence rate for all patients were 44.0% and 42.0%, respectively. Four patients died from non-HCC recurrence post-LT (one from cardiac infarction; one from community acquired pneumonia; and two from chronic rejection post-LT).

 

The 104 samples of HCC with HBV-associated cirrhosis were stained immunohistochemically. GPC-3 expression was detected in 71.2% (74/104) of the tumor specimens. In most GPC-3-positive cases, GPC-3 staining occurred in the cytoplasm of cancerous cells. GPC-3 was sometimes expressed on the cell membrane surface. Adjacent non-cancerous cells were negative. As shown in Table 1, GPC-3 expression was positively correlated with moderate and poor histological tumor differentiation, large tumor size, post-operative recurrence, non-intact encapsulation, portal vein invasion, greater pathological stage and Milan criteria (all P<0.05). Staining was not correlated with age, gender, pre-operative AFP, Child-Pugh score, number of tumors, MELD score, or BMI (all P>0.05).

 

Univariate and multivariate analyses showed an association between GPC-3 expression and survival duration and recurrence in HCC patients with HBV-associated cirrhosis after LT. The univariate analysis had a stepwise inclusion and indicated that moderate and poor differentiation, pre-operative AFP ≥20 ng/mL, GPC-3-positive expression, total diameter of tumor nodules ≥8 cm, portal vein invasion, beyond Milan criteria and high TNM stages were significant prognostic variables for cumulative shorter survival and briefer disease-free survival (DFS) rates (all P<0.05, Table 2). In multivariate analysis, worse tumor differentiation (P=0.004), GPC-3-positive expression (P=0.031), beyond Milan criteria (P=0.029), and pre-operative AFP ≥20 ng/mL (P=0.023) were independent risk factors related to 5-year overall survival. The independent risk factors including worse tumor differentiation (P=0.016), pre-operative AFP ≥20 ng/mL (P=0.030), portal vein invasion (P=0.025), beyond Milan criteria (P=0.002) and GPC-3-positive expression (P=0.047) were correlated with tumor recurrence after LT (Table 3).

 

To assess the prognostic value of GPC-3, the cumulative survival and tumor recurrence rates of post-LT GPC-3-postive and -negative patients were compared. The survival rate of GPC-3-positive patients was lower than that of GPC-3-negative patients (38.2% vs 75.4%, P<0.001; Table 2). After LT, the cumulative DFS rate of GPC-3-negative patients was higher than that of GPC-3-positive patients (69.7% vs 30.8%, P=0.001) (Fig. 2). The overall survival time of GPC-3-negative patients was greater than that of GPC-3-positive patients (101.89±8.62 vs 52.98±5.76 months, P<0.001). The prognostic value of GPC-3 was tested further in 3 patient subtypes: those who met the Milan criteria, those with pre-operative AFP ≥20 ng/mL, and those with well-differentiated HCC. These categories were independent prognostic factors for survival and recurrence. Among these subtypes, patients with GPC-3-positive expression had lower survival rates and briefer DFS rates than patients with GPC-3-negative expression except in the patient subgroup whose DFS rates met the Milan criteria and these patients were not different (Figs. 3-5).

 

 

The most common cause of HCC in China is HBV infection, which frequently also causes cirrhosis. Hepatectomy is the best treatment for patients with HCC. However, hepatectomy is not feasible for those whose tumor has aggressive behavior, for patients whose liver function is poor and for those who have multifocal HCC. Thus, LT is a unique option for these patients. LT can offer good outcomes in well-selected candidates with a 5-year survival rate ranging from 53% to 80%, even when vascular and hilar lymph nodes are involved.^[24] The Milan, Barcelona, and UCSF criteria allow precise selection of HCC patients capable of undergoing LT without a high risk of tumor recurrence. Also, efforts have been made to expand the criteria for LT, including the Hangzhou criteria^[25] which include total tumor size ≤8 cm with pre-operative AFP ≤400 ng/mL and moderate-to-well differentiation. These criteria focus not only on the size and number of tumors but also on serum AFP and histological differentiation. Since patients' prognosis is so important that scientists have been trying to find the prognostic factors, GPC-3 is one of them. GPC-3 is closely correlated with the prognosis of patients with HCC after LT. This retrospective analysis revealed that 71.2% of patients with HBV-associated HCC were GPC-3-positive. The clinicopathological parameters correlated with GPC-3-positive included tumor size, intact encapsulation, portal vein invasion, histological differentiation and the Milan criteria. After LT, GPC-3-positive patients had worse prognosis, as indicated by univariate and Kaplan-Meier survival analyses. Multivariate Cox-regression analysis indicated differences as well. GPC-3 expression was found to be an independent risk factor for mortality rate and recurrence with the Milan criteria, differentiation and pre-operative AFP.

 

GPC-3 has been used for HCC diagnosis. Furthermore, GPC-3-positive indicated poor prognosis in HCC patients who underwent hepatectomy.^{[17, 18]} However, whether GPC-3 plays the predictive prognostic role in LT is unclear, especially for HCC patients with HBV-associated cirrhosis. The present study demonstrated that GPC-3-positive expression may be a biomarker for poor prognosis in HBV-associated HCC patients who underwent LT, similar to the Milan criteria, differentiation and pre-operative AFP.

 

The Milan criteria are widely accepted for selecting HCC patients waiting for LT.^[26] Serum AFP is also widely used as a biological biomarker for diagnosing and monitoring HCC and post-operative recurrence. Here, the cutoff value was set to 20 ng/mL (AFP was divided into <20 ng/mL, 20-400 ng/mL, and ≥400 ng/mL, and the latter two groups were not statistically different, so they were considered to be one group). Pre-operative AFP ≥20 ng/mL was an independent factor for poor prognosis and recurrence. In the pre-operative AFP ≥20 ng/mL group, GPC-3-negative patients had greater 5-year survival and DFS rates than GPC-3-positive patients. However, in the pre-operative AFP <20 ng/mL group, there was no statistically significant difference. Half of the HCC patients had abnormal pre-operative serum AFP^[27] suggesting that GPC-3 might be a useful prognostic biomarker when used with dynamic monitoring of post-operative serum AFP in this subgroup.

 

The Milan criteria recommend transplantation for patients with up to three nodules with a maximum diameter of 3 cm or a single nodule with a maximal diameter of 5 cm is still the current selection standard.^[28] Our cohort showed that the Milan criteria was still the most important factor associated with survival and DFS (odds ratio 2.947 and 4.161). The expansion of criteria for LT in multiple centers has led to the introduction of histological features (i.e. vascular invasion, tumor differentiation, satellite nodules) in the prediction of the risk of recurrence after transplantation.^{[26, 29, 30]} We found that the survival time of patients who met the Milan criteria and were GPC-3-positive was shorter than that of those who met the Milan criteria but were GPC-3-negative. However, GPC-3 status did not affect DFS rates, indicating that GPC-3 has limited ability to predict tumor recurrence in patients who met the Milan criteria. No differences were detected in groups who were not evaluated by the Milan criteria. In this study, GPC-3 expression was correlated with the poor or moderate differentiation of tumor. The predictive value of tumor differentiation for HCC after LT has been reported in some studies.^{[21, 31]} Our univariate survival and multivariate Cox-regression analyses revealed that well-differentiated HCC had better survival and DFS rate. GPC-3 expression was positively correlated with lower grade tumor differentiation. The prognosis of GPC-3-negative patients in the well-differentiated group was better than that of GPC-3-positive ones.

 

The above results proved that GPC-3 was a useful marker of prognosis and recurrence in HCC patients with HBV-associated cirrhosis who had undergone LT. Thus, GPC-3 may be a valuable marker for speculating other clinicopathological parameters such as differentiation, tumor size and TNM stage, etc. GPC-3 is a potential component in future selection criteria of LT in patients with HBV-associated HCC.

 

 

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