Serum Golgi protein 73 and glypican-3: early diagnostic biomarkers for hepatocellular carcinoma

Doaa F Gad; Islam Eid Mohamed; Essam Elmahdi; Abdelnaser A Badawy; Gamal Othman; Mohamed G Dawoud; Mohammed Diasty; Maysaa E Zaki; Mohamed M Abd El-Mawgod

doi:10.4103/kamj.kamj_24_19

ORIGINAL ARTICLE

Year : 2021 | Volume : 27 | Issue : 3 | Page : 51-57

Serum Golgi protein 73 and glypican-3: early diagnostic biomarkers for hepatocellular carcinoma

Doaa F Gad¹, Islam Eid Mohamed¹, Essam Elmahdi¹, Abdelnaser A Badawy², Gamal Othman², Mohamed G Dawoud³, Mohammed Diasty⁴, Maysaa E Zaki⁵, Mohamed M Abd El-Mawgod⁶
¹ Department of Internal Medicine, Faculty of Medicine, Mansoura University, Mansoura, Egypt
² Department of Medical Biochemistry, Faculty of Medicine, Mansoura University, Egypt
³ Department of General Surgery, Gastrointestinal Surgery Center, Faculty of Medicine, Mansoura University, Mansoura, Egypt
⁴ Department Tropical Medicine, Faculty of Medicine, Mansoura University, Mansoura, Egypt
⁵ Department of Clinical Pathology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
⁶ Department of Public Health and Community Medicine, Faculty of Medicine, Al-Azhar University, Assiut, Egypt

Date of Submission	18-Sep-2019
Date of Decision	25-Sep-2020
Date of Acceptance	25-Nov-2019
Date of Web Publication	19-Aug-2022

Correspondence Address:
MD Essam Elmahdi
Department of Internal Medicine, Faculty of Medicine, Mansoura University, Mansoura
Egypt

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/kamj.kamj_24_19

Abstract

Objectives The prognosis of hepatocellular carcinoma (HCC) is extremely worse, and chronic hepatitis C virus infection is one of the most important causes of HCC. The use of diagnostic serological markers in following up the high-risk individuals for developing HCC may help in its early detection and therapy. This study aimed to assess the effectiveness of serum Golgi protein 73 (GP73) and glypican-3 (GPC3) as new tumor biomarkers for early detection of HCC.
Patients and methods A case–control study included 125 patients infected with hepatitis C virus, comprising 60 with cirrhosis and 65 with HCC, in addition to 60 healthy individuals considered as the normal control group. Serum levels of GP73, GPC3, and alpha-fetoprotein (AFP) were assessed by enzyme-linked immunosorbent assay technique.
Result Serum GP73 and AFP levels were significantly higher in patients with HCC as compared with cirrhosis and control groups; however, their levels were significantly increased in patients with cirrhosis as compared with the healthy group (both P<0.001). However, GP73 was more sensitive than AFP in the diagnosis of HCC as the area under the receiver operating characteristic curve (area under the curve) with 95% confidence interval was 0.98 (0.95–1.0) for GP73 and 0.82 (0.74–0.90) for AFP. However, the area under the curve with 95% confidence interval for GPC3 was 0.44 (0.36–0.53). There was a significant association between AFP level and Barcelona clinic liver cancer staging of HCC (P=0.02). In addition, the serum level of GPC3 was higher in cirrhotic patients than other groups (P=0.007).
Conclusion Serum GP73 and not GPC3 can be used as early potential tumor biomarker for HCC diagnosis and also to differentiate HCC from cirrhosis.

Keywords: alpha-fetoprotein, cirrhosis, glypican-3, Golgi protein 73, hepatocellular carcinoma, hepatitis C virus

How to cite this article:
Gad DF, Mohamed IE, Elmahdi E, Badawy AA, Othman G, Dawoud MG, Diasty M, Zaki ME, Abd El-Mawgod MM. Serum Golgi protein 73 and glypican-3: early diagnostic biomarkers for hepatocellular carcinoma. Kasr Al Ainy Med J 2021;27:51-7

How to cite this URL:
Gad DF, Mohamed IE, Elmahdi E, Badawy AA, Othman G, Dawoud MG, Diasty M, Zaki ME, Abd El-Mawgod MM. Serum Golgi protein 73 and glypican-3: early diagnostic biomarkers for hepatocellular carcinoma. Kasr Al Ainy Med J [serial online] 2021 [cited 2023 Dec 5];27:51-7. Available from: http://www.kamj.eg.net/text.asp?2021/27/3/51/354080

Introduction

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most common reason for cancer-induced mortality in the world [1]. It was noticed that hepatitis C virus (HCV) infection presents in ~40–80% of patients with HCC [2]. HCC is characterized by high mortality with decreased 5-year survival rate as most of the cases are diagnosed in advanced stage, a problem making therapy ineffective [3]. Early diagnosis of HCC makes effective treatment by either surgical resection of the tumor or liver transplantation [4]. Early detection and intervention of HCC improve its prognosis; hence, the use of early detection serological markers in high susceptible patients decreases HCC mortality [4]. The routine investigations used for early discovery of HCC were serum alpha-fetoprotein (AFP) level and abdominal ultrasound. During the past four decades, AFP was the only used marker for HCC with 80–94% specificity and 41â 65% sensitivity [5].

Many HCC cases were diagnosed without increased AFP level. However, other cases showed elevation only when tumor size is more than 3–5 cm [6]. This controversy makes AFP unsuitable marker for HCC. Therefore, the identification of highly sensitive and more specific marker than AFP is urgently needed for early detection of HCC [7],[8]. Many noninvasive serum markers such as Golgi protein 73 (GP73) and glypican- 3 (GPC3) have been specified for HCC [9].

GP73, named additionally as Golgi phospho- protein 2, is a 73-kDa protein composed of 400 amino acids. Type II Golgi membrane protein contains a transmembrane domain, a short N- terminal cytoplasmic domain, and a larger lu- minal C-terminal domain [10]. The C-terminal ectodomain is the main functional domain of GP73 and secreted and can be detected in the blood [11]. The hepatic expression of GP73 is very low in nor- mal adults, and it is mainly produced by the biliary cells not hepatic cells [12]. The hepatic expression of GP73 is highly upregulated in patients with liver diseases [13]. HCV infection causes an increase in the GP73 level, which in turn upregulates apopro- tein E, which enhances and stimulates HCV secre- tion [14]. The hepatic expression of GP73 is gradually increased from healthy liver and hepatitis progressing to liver cancer [15].

GPC3 is a cell-surface 60-kDa protein, related to heparin-sulfate proteoglycans family [16], which contains GPC1 to GPC6. GPC3 is cut at Arg358 and Ser359 to produce amino (N) terminal 40-kDa protein and carboxyl (C) terminal 30-kDa membrane- bound protein. The N-terminal protein of GPC3, named also soluble GPC3, is released in the serum and can be used as a marker in diagnosis of patients with HCC [17]. GPC3 has an integral role in cell proliferation and tumor suppression [18]. GPC3 is expressed normally in fetal hepatic tissues and placenta with very minimal expression in hepatic tissues of adults [16]. Patients with HCC had high level of serum GPC3 in comparison with healthy individuals and chronic hepatitis patients, a result making serum GPC3 a possible specific biomarker for HCC [19]. The aim of the current study was to assess the efficiency of GP73 and GPC3 as tumor biomarkers for HCC detection.

Patients and methods

A case–control study was conducted on 125 patients infected with HCV, comprising 60 patients with cirrhosis and 65 pa- tients with HCC. Patients were recruited from specialized medical hospital, Mansoura University from February 2018 to February 2019. The AQ6 patients had positiveHCV an- tibodies (Access Bio-RadCo., France,3 BOULEVARD RAYMOND POINCARE 92430) and were con?rmed by quantitative viral RNA detection using Taq Man HCV quantitative test version 2.0 (Roche Molecular Diagnostic, Branchburg, New Jersey, USA) according to guidance of manufacturer. Exclusion criteria included individuals with HBV and with other cancers. All patients were diagnosed by clinical, laboratory, and radiological examinations including abdominal ultrasound and computed tomography. Patients were further classified according to histopathological grading, tumor size, Child–Pugh class system, and Barcelona clinic liver cancer (BCLC) staging system [20]. In addition to the patients, the study included 60 healthy individuals as a control group. The research was approved by Mansoura University ethical committee (Code: R.19.05.498), and approval consent was obtained from each participant.

Ten milliliters of blood was withdrawn from each participant and divided into two parts; the first part was collected into citrated tube for complete blood count and the third part into plain tube, and sera were separated and subjected to the analysis for complete liver functions and the determination of AFP by enzyme-linked immunosorbent assay (Catalog #: EIA-1468; ELISA-DRG International Inc., USA). The DRG AFP ELISA kit is a solid-phase ELISA based on the sandwich principle. Moreover, sera were subjected to the determination of GP73 and GPC3 by ELISA technique.

Serum Golgi protein 73 by sandwich-based enzyme- linked immunosorbent assay method

Serum GP73 level was assayed by sandwich-based ELISA method (RayBiotech GP73 Quantitative Detection Kit, Catalog #: ELISA-GP73-96; RayBiotech, Norcross, Georgia, USA) according to instructions of the manufacturer. A specific anti- human GP73 monoclonal antibody is immobilized on the surface of a 96-well plate. Standards and samples were added into the wells of the plate. After incubation, a secondary detection antibody was added. A second incubation was done, then an enzymatic conjugate was added that can bind antibody bound to the plate to create a complex of solid-phase antibody–antigen–antibody. After addition of a coloring substrate, this enzyme reacts to produce a color relative to GP73 concentration in the sample. Finally, the OD of the wells at 450 nm was read on a spectrophotometric plate reader. The concentration was expressed as ng/ml.

Glypican-3 by quantitative sandwich enzyme immunoassay

This assay employs the quantitative sandwich enzyme immunoassay technique (Catalog Number DGLY30; RD-Quantikine 614 McKinley Place NE, Minneapolis, Minnesota, USA) according to the manufacturer's instructions. Human GPC3-specific monoclonal antibody had been precoated onto the surface of a 96-well microplate. Samples as well as standards were added to the wells, and any GPC3 substrate was bound to the fixed antibody. Washing of any free substrates was done, and then human GPC3-specific enzyme-linked polyclonal antibody was added to the wells. Washing was done again to remove any free antibody enzyme reagent, and then substrate was added to the wells to give color, which is directly proportional to GPC3 concentration. The color development was stopped, and the intensity of the color was measured. Finally, the OD of the wells at 450 nm was read on a spectrophotometric plate reader. A total of 27 assays were evaluated and the minimum detectable dose of human GPC3 ranged from 3.07 to 20.6 pg/ml. The mean minimum detectable dose was 9.18 pg/ml.

Statistical analysis

Data were analyzed using (SPSSsoftware,version22.0. Chicago, IL, USA). Quantitative nonparametric data were analyzed by Kruskal–Wallis test to compare more than two independent groups. Quantitative parametric data were assessed by one-way analysis of variance test to compare more than two independent groups. Receiver operating characteristic (ROC) curve analysis was test to discriminate diseased cases from nondiseased cases. Significance of the obtained results was judged at the (0.05) level.

Results

Our study found that HCC occurred at a relatively younger age in comparison with the cirrhotic group (50±5.2 and 55.6±9.4, respectively) and more in males than females as compared with other groups. There were significant elevations of alanine transferase, aspartate transferase, and total bilirubin; however, there was a significant decrease in white blood cells and platelets levels in HCC group as compared with other groups. Regarding tumor markers, there was a significant elevation of AFP and GP73 in HCC group in comparison with the other groups. Moreover, there was a significant elevation of AFP and GP73 in cirrhosis group in comparison with the controls. However, there is notable elevation of GPC3 in cirrhotic group in comparison with the other groups. The cancer grading in the HCC group revealed that most of patients with HCC were grade 2 (38%) and BCLC stage A (35%), tumor size more than 5 cm (43%) and most of them were Child–Pugh classes B and C (42 and 46%). Data are shown in [Table 1].

Table 1 Demographic and laboratory data in studied groups

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[Table 2] shows the relationship between the levels of tumor markers and the tumor grading among HCC group. There was no association between tumor markers and any of the tumor grade among HCC group, except that there was a significant association between AFP level and BCLC staging (P=0.01). In addition, GPC3 was increased in Child–Pugh class C than other groups (P=0.009).

Table 2 Serum levels of alpha-fetoprotein, Golgi protein 73, and glypican-3 in different hepatocellular carcinoma grades

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ROC curve of the efficiency of serum AFP, GP73, and GPC3 in the diagnosis of HCC from the whole study population showed that GP73 was more sensitive and specific than other tumor markers in the diagnosis of HCC, as shown in [Figure 1]. The area under the receiver operating characteristic (AUROC) with 95% confidence interval (CI) for GP73 was 0.98 (0.95–1.0), with 98% sensitivity and 98% specificity at an optimal cutoff value of 265 ng/ml. The AUROC for AFP with 95% CI was 0.82 (0.74–0.90), with 76% sensitivity and 87% specificity at a cutoff of 9.4 ng/ml. The AUROC with 95% CI for GPC3 was 0.45 (0.36–0.53), with 63% sensitivity and 34% specificity at a cutoff value of 5.8 pg/ml.

Figure 1 Receiver operating characteristic curve of the efficiency of serum AFP, GP73, and GPC3 in diagnosis of HCC from the whole study population. AFP, alpha-fetoprotein; GP73, Golgi protein 73; GPC3, glypican-3; HCC, hepatocellular carcinoma.

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ROC curve of the efficiency of serum AFP, GP73, and GPC3 in diagnosis of HCC from cirrhosis group revealed that GP73 has higher sensitivity and specificity than AFP, as shown in [Figure 2]. The AUROC with 95% CI for GP73 was 0.96 (0.91–1.0), with 97% sensitivity and 95% specificity at an optimal cutoff value of 273.5 ng/ml. The AUROC with 95% CI for AFP was 0.80 (0.71–0.88), with sensitivity of 73% and specificity of 84% at a cutoff value of 10.3 ng/ml. The AUROC with 95% CI for GPC3 was 0.41 (0.31–0.52), with 44% sensitivity and 47%specificity at a cut-off value of 6.9 pg/ml.

Figure 2 Receiver operating characteristic curve of the efficiency of serum AFP, GP73, and GPC3 in differentiating HCC from cirrhosis. AFP, alpha-fetoprotein; GP73, Golgi protein 73; GPC3, glypican-3; HCC, hepatocellular carcinoma.

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Discussion

Chronic HCV infection is considered a fundamental cause of HCC, which is the second most prevalent cause of cancer mortality [21]. It was reported that GP73 was first discovered in biliary cells in patients with acute hepatitis, cirrhosis, and HCC [22]. GP73 is considered a possible biomarker for early HCC diagnosis as it can differentiate HCC cases from cirrhosis [23]. Our findings demonstrated that the serum level of GP73 in HCC group was higher (288±7.8 ng/ml) than those in cirrhosis (230 ±27.8 ng/ml) and normal control group (135±9.7 ng/ ml), a result explained by the secretion of GP73 protein by hepatoma cells into the circulation in first stage, resulting in the significantly elevated serum GP73 in HCC [24]. Results were in agreement with Anand et al. [25], who found that increased hepatic expression of GP73 in chronic liver disease, including hepatitis, cirrhosis, and HCC.

Comparing GP73 and AFP as tumor markers in diagnosis of HCC and in differentiating HCC from cirrhosis, our study has shown that GP73 is a good specific marker than AFP in HCC diagnosis, as AUROC for GP73 was 0.98 whereas for AFP was 0.82. This result is in agreement with Hu et al. [26], Tian et al. [27], and Mariam et al. [28] who reported that serum level of GP73 is highly increased in patients with HCC as compared with either patients with nonmalignant liver diseases or the healthy individuals, and has a good diagnostic value than AFP for diagnosis of HCC, but not in agreement with Ozkan et al. [29], who reported that GP73 has a low value for the diagnosis and prognosis of early HCC, whereas AFP was better. These differences might be owing to different population characteristics, including the causes of cancer, ethnicity, income level, and detection methods used. However, the use of GP73 to replace AFP or the combination of both parameters in HCC detection should be further evaluated. Previous studies examined the relation between both GP73 and AFP markers and severity of HCC, but results are still controversial. Our study demonstrated that there is no association between tumor markers and any of the tumor characteristics among HCC group, except that, there was a significant association between AFP level and BCLC staging. Hu et al. [26] showed that GP73 had a good ability to differentiate cirrhosis from stages I and II PHC. In addition, serum GP73 and AFP levels were significantly higher in patients with stages III and IV cancer compared with stages I and II [30].

GPC3, one of glypican family, is a second protein marker that inhibits cell proliferation and causes apoptosis and is found to be variable in different types of cancers [18]. Previous studies have proposed that serum GPC3 is a specific marker for HCC [19]. Our results revealed that there was a significant elevation of GPC3 in cirrhotic group in comparison with HCC and normal control groups (8.43, 6.75, and 6.98 ng/ml, respectively), and there was no differences between HCC and normal group. In addition, the AUROC for GPC3 was 0.43 (95% CI, 0.33–0.52), with a sensitivity of 58% and a specificity of 34% at a cut-off value of 5.8 ng/ml, indicating that GPC3 has no role in HCC diagnosis. Some studies have found lower or equivalent serum levels of GPC3 in patients with HCC compared with patients with liver cirrhosis [29],[31]. Another study showed that serum GPC3 is elevated in patients with HCC as compared with healthy individuals and hepatitis or liver cirrhotic patients [32]. Liu et al. [33] showed that GPC3 has higher diagnostic value in patients with HCC and liver cirrhosis, with sensitivity of 56% and specificity of 89%, and the AUROC curve was 0.88, indicating that GPC3 has a good diagnostic value. The differences in the results owing to different characteristics of HCC with many underlying risk factors make serum GPC3 a possible suitable marker for HCC in one population but not in the other.

Conclusion

In conclusion, our study strongly observed that GP73, not GPC3, is a potential tumor biomarker for HCC. The GP73 is superior to AFP in HCC diagnosis, and to establish the use of GP73 as a tumor marker for HCC diagnosis, future studies on a large sample should be done.

Financial support and sponsorship

Nil.

Conflicts of interest

Authors declare no conflicts of interest

References

1.	Jemal A, Bray F, Center MM. Global cancer statistics. Cancer J Clin 2011; 61:69–90.
2.	Mittal S, El-Serag HB. Epidemiology of hepatocellular carcinoma: consider the population. J Clin Gastroenterol 2013; 47:S2–S6.
3.	Schutte K, Bornschein J, Malfertheiner P. Hepatocellular carcinoma: epidemiological trends and risk factors. Dig Dis 2009; 27:80–92.
4.	El-Serag HB, Mason AC, Key C. Trends in survival of patients withhepatocellular carcinoma between 1977 and 1996 in the United States. Hepatology 2001; 33:62–65.
5.	Debruyne EN, Delanghe JR. Diagnosing and monitoring hepatocellularcarcinoma with alpha-fetoprotein: New aspects and applications. Clin Chim Acta 2008; 395:19–26.
6.	Flores A, Marrero JA. Emerging trends in hepatocellular carcinoma: focus on diagnosis and therapeutics. Clin Med Insights Oncol 2014; 8:71–76.
7.	Gish R. Early detection of hepatocellular carcinoma through surveillance using biomarkers. Gastroenterol Hepatol 2014; 10:121–123.
8.	Shaker MK, Abdel Fattah HI, Sabbour GS. Annexin A2 as a biomarker for hepatocellular carcinoma in Egyptian patients. World J Hepatol 2017; 9:469–476.
9.	Masuzaki R, Karp SJ, Omata M. New serummarkers ofhepatocellular carcinoma. Semin Oncol 2012; 39:434–439.
10.	Kim HJ, Lv D, Zhang Y, Peng T, Ma X. Golgi phosphoprotein 2 inphysiology and in diseases. Cell Biosci 2012; 2:31.
11.	Bachert C, Fimmel C, Linstedt AD. Endosomal trafficking and proprotein convertase cleavage of cis Golgi protein GP73 produces marker for hepatocellular carcinoma. Traffic 2007; 8:1415–1423.
12.	Ba MC, Long H, Tang YQ, Cui SZ. GP73 ex-pression and its significance in the diagnosis of hepatocellular carcinoma: a review. Int J Clin Exp Pathol 2012; 5:874–881.
13.	Gu Y, Chen W, Zhao Y, Chen L, Peng T. Quantitative analysis of elevated serum Golgi protein-73 expression in patients with liver diseases. Ann Clin Biochem 2009; 46:38–43.
14.	Hu L, Yao W, Wang F, Rong X, Peng T. GP73 is upregulated by hepatitis C virus (HCV) infection and enhances HCV secretion. PLOS One 2014; 9:e90553.
15.	Shan SG, Gao YT, Xu YJ. Gradually increased Golgi protein 73 expression in the progression of benign liver diseases to precancerous lesions and hepatocellular carcinoma correlates with prognosis of patients. Hepatol Res 2013; 43:1199–1210.
16.	Jia HL, Ye QH, Qin LX, Budhu A, Forgues M, Chen Y, et al. Gene expression profiling reveals potential biomarkers of human hepatocellular carcinoma. Clin Cancer Res 2007; 13:1133–1139.
17.	Ho M, Kim H. Glypican-3: a new target for cancer immunotherapy. Eur J Cancer 2011; 47:333–338.
18.	Filmus J. The contribution of in vivo manipulation of gene expression to the understanding of the function of glypicans. Glycoconj J 2002; 19:319–323.
19.	Yu JP, Xu XG, Ma RJ. Development of a clinicalchemiluminescent immunoassay for serum GPC3 and simultaneousmeasurements alone with AFP and CK19 in diagnosis of hepatocellular carcinoma. J Clin Lab Anal 2015; 29:85–93.
20.	Bruix J, Sherman M. Management of hepatocellular carcinoma: an update. Hepatology 2011; 53:1020–1022.
21.	Ryerson AB, Eheman CR, Altekruse SF. Annual report to the nation on the status of cancer, 1975-2012, featuring the increasing incidence of liver cancer. Cancer 2016; 122:1312–1337.
22.	Marrero J, Romano P. GP73, a resident Golgi glycoprotein, is a novel serum marker for Hepatocellular carcinoma. J Hepatol 2005; 43:1007–1012.
23.	Comunale MA, Lowman. M, Long RE. Proteomic analysis of serum associated fucosylated glycoproteins in the development of primary hepatocellular carcinoma. J Proteome Res 2006; 6:308–315.
24.	Farag RMA, Al Ayobi D, Alsaleh KA, Kwon H, EL-Ansary A, Dawoud EA. Studying the impact of Golgi protein 73 serving as a candidate biomarker in early diagnosis for hepatocellular carcinoma among Saudi patients. Asian Pac J Cancer Prev 2019; 20:215–220.
25.	Anand M, Harmin H, Timothy B. Glycosylation and liver cancer. Adv Cancer Res 2015; 126:257–279.
26.	Hu JS, Wu DW, Liang S, Miao XY. GP73, a resident Golgi glycoprotein, is sensibility and specificity for hepatocellular carcinoma of diagnosis in a hepatitis B-endemic Asian population. Med Oncol 2010; 27:339–345.
27.	Tian L, Wang Y, Xu D, Gui J, Jia X, Tong H, et al. Serological AFP/Golgi protein 73 could be a new diagnostic parameter of hepatic diseases. Int J Cancer 2011; 129:1923–1931.
28.	Mariam A, Emad F, Dalia WF, Elela HA, Abdelsalam OH. Golgi protein 73 versus alpha fetoprotein as a marker for hepatocellular carcinoma. Medicine (Baltimore) 2015; 94:e659.
29.	Ozkan H, Erdal H, Tutkak H, Karaeren Z, Yakut M, Yuksel O. Diagnostic and prognostic validity of Golgi protein 73 in hepatocellularcarcinoma. Digestion 2011; 83:83–88.
30.	Sun Y, Yang H, Mao Y, Xu H, Zhang J, Li G, et al. Increased Golgi protein 73 expression in he-patocellular carcinoma tissue correlates with tumor aggression but not survival. J Gastroenterol Hepatol 2011; 26:1207–1212.
31.	Wang Y, Yang H, Xu H. Golgi protein 73, not Glypican-3, may be a tumor marker complementary to alpha fetoprotein forhepatocellular carcinoma diagnosis. J Gastroenterol Hepatol 2014; 29: 597–602.
32.	Lee HJ, Yeon JE, Suh SJ. Clinical utility of plasmaglypican-3 and osteopontin as biomarkers of hepatocellular carcinoma. Gut Liver 2014; 8:177–185.
33.	Liu JW, Zuo XL, Wang S. Diagnosis accuracy of serum glypican-3 level in patients with hepatocellular carcinoma and liver cirrhosis: a meta-analysis. Eur Rev Med Pharmacol Sci 2015; 19:3655–3673.