Clinical application of specific antibody against glypican-3 for hepatocellular carcinoma diagnosis

Glypican-3(GPC3) is a promising tumor marker for hepatocellular carcinoma(HCC) diagnosis with high sensitivity and specificity.The aim of this study was to establish an immunohistochemical detection method for GPC3 using the 7D11 monoclonal antibody(7D11 mAb) and evaluate its application for HCC diagnosis.The feasibility of the 7D11 mAb was evaluated by immunohistochemistry performed on adjacent normal liver and intrahepatic cholangiocarcinoma(ICC) samples,Furthermore,the serum GPC3 levels were evaluated in 40 HCC patients,7 ICC patients and 50 healthy donors.The results showed that GPC3 was expressed in 85% of HCC tissues(34/40),but was undetectable in ICC tissues and adjacent normal tissues.GPC3 was significantly increased in the serum of HCC patients(17/40,42.5%) but was undetectable in the serum of ICC patients(0/7,0%) and healthy donors(0/50,0%).This prospective study evaluated the clinical usefulness of 7D11 mAb for GPC3 detection in HCC patients.In conclusion,the use of 7D11 mAb might be good for GPC3 large-scale applications for clinical diagnosis of HCC.     

GPC3-targeted CAR-T cells secreting B7H3-targeted BiTE exhibit potent cytotoxicity activity against hepatocellular carcinoma cell in the in vitro assay

Hepatocellular carcinoma (HCC), the most common primary liver cancer has a high mortality in China, and it is usually diagnosed at a late stage, thereby leaving patients with few effective treatment options. Chimeric antigen receptor-T (CAR-T) cell therapy, a novel immunotherapy that has shown promising results in leukemia, lymphoma and multiple myeloma, is also expected to work well in solid tumors, including HCC. However, the ideal therapeutic efficacy has not yet been achieved, in part due to tumor antigen escape caused by antigen heterogeneity. To overcome such challenge, we screened a panel of biomarkers in HCC cell lines and found that GPC3 and B7H3 were highly expressed on HCC with expression heterogeneity. Then we developed a novel bispecific T cell engagers CAR-T (CAR.T-BiTEs) that drives the expression of a CAR specific for GPC3 and BiTEs against CD3 and B7H3, herein referred to as “GPC3-BiTE CAR.” We found that BiTEs promoted the increased activation of untransduced T cells and IFN-γ release. Moreover, BiTEs secreted by GPC3-BiTE CAR-HEK293T cells promoted increased cytotoxicity activity of untransduced T cells against GPC3+/B7H3+ (GPC3 positive/B7H3 positive) and GPC3-/B7H3+(GPC3 negative/B7H3 positive) HCC cell lines. In vitro function assays showed that GPC3-BiTE CAR-T cells exhibited greater cytotoxicity activity against GPC3+/B7H3+ HCC cell lines than GPC3 CAR-T cells (GPC3-targeted CAR-T cells) and B7H3 CAR-T cells (B7H3-targeted CAR-T cells). Furthermore, GPC3-BiTE CAR-T cells exhibited superior cytotoxicity against GPC3 negative HCC cell lines compared with GPC3 CAR T cells. In conclusion, our study showed that GPC3-BiTE CAR T cells exhibited superior antitumor activity than single-target CAR-T cells and can overcome tumor escape induced by antigen heterogeneity, suggesting that this could be a promising therapeutic strategy for HCC.     

Glypican-3, a novel prognostic marker of hepatocellular cancer, is related with postoperative metastasis and recurrence in hepatocellular cancer patients

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 (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.     

A distinct repertoire of autoantibodies in hepatocellular carcinoma identified by proteomic analysis

Chronic infections with hepatitis B (HBV) and hepatitis C (HCV) viruses are major risk factors for hepatocellular carcinoma (HCC). We have utilized a proteomic approach to determine whether a distinct repertoire of autoantibodies can be identified in HCC. Sera from 37 patients with HCC and 31 subjects chronically infected with HBV or HCV without HCC were investigated. Sera from 116 patients with other cancers, three patients with systemic lupus erythematosus, and 24 healthy subjects were utilized as controls. We report the identification of eight proteins, for each of which autoantibodies were detected in sera from more than 10% of patients with HCC but not in sera from healthy individuals (p < 0.05). Autoantibodies to four of these proteins were detected at a comparable frequency in sera from patients with chronic hepatitis. The other four proteins, which consisted of calreticulin isoforms, cytokeratin 8, nucleoside diphosphate kinase A, and F(1)-ATP synthase beta-subunit, induced autoantibodies among patients with HCC, independently of their HBV/HCV status. Calreticulin, and a novel truncated form of calreticulin (Crt32) we have identified, most commonly elicited autoantibodies among patients with HCC (27%). We conclude that a distinct repertoire of autoantibodies is associated with HCC that may have utility in early diagnosis of HCC among high risk subjects with chronic hepatitis.     

Human Monoclonal Antibody Targeting the Heparan Sulfate Chains of Glypican-3 Inhibits HGF-Mediated Migration and Motility of Hepatocellular Carcinoma Cells

Heparan sulfate proteoglycans (HSPGs) participate in many processes related to tumor development, including tumorigenesis and metastasis. HSPGs contain one or more heparan sulfate (HS) chains that are covalently linked to a core protein. Glypican-3 (GPC3) is a cell surface-associated HSPG that is highly expressed in hepatocellular carcinoma (HCC). GPC3 is involved in Wnt3a-dependent HCC cell proliferation. Our previous study reported that HS20, a human monoclonal antibody targeting the HS chains on GPC3, inhibited Wnt3a/β-catenin activation. In the current study, we showed that the HS chains of GPC3 could mediate HCC cells’ migration and motility. Knocking down GPC3 or targeting the HS chains by HS20 inhibited HCC cell migration and motility. However, HS20 had no effect on GPC3 knockdown cells or GPC3 negative cells. In addition, an antibody that recognizes the core protein of GPC3 did not change the rate of cell motility. HCC cell migration and motility did not respond to either canonical or non-canonical Wnt induction, but did increase under hepatocyte growth factor (HGF) treatment. HS20-treated HCC cells exhibited less ability for HGF-mediated migration and motility. Furthermore, HS20 inhibited in vitro HCC spheroid formation and liver tumor growth in mice. GPC3 interacted with HGF; however, a mutant GPC3 lacking the HS chain showed less interaction with HGF. Blocking the HS chains on GPC3 with HS20 reduced c-Met activation in HGF-treated HCC cells and 3D-cultured spheroids. Taken together, our study suggests that GPC3 is involved in HCC cell migration and motility through HS chain-mediated cooperation with the HGF/Met pathway, showing how HS targeting has potential therapeutic implications for liver cancer.     

Identification of complement C3a as a candidate biomarker in human chronic hepatitis C and HCV-related hepatocellular carcinoma using a proteomics approach

Although the significant risk factors for hepatocellular carcinoma (HCC) are well known from epidemiological studies, diagnosis of this disease at an early stage is difficult, and HCC remains one of the leading causes of cancer death worldwide. Thus, to identify any useful HCC-related biomarkers is still a need. We performed SELDI-TOF MS to identify differentially expressed proteins in HCC serum using weak cation exchange protein chips. Protein characterization was performed by 2-DE separation and nano flow LC-MS/MS. A total of 55 sera were collected from HCC patients and compared with those from 48 patients with chronic hepatitis and 9 healthy individuals. A candidate marker of about 8900 Da was detected as differentially expressed in patients with chronic hepatitis C and hepatitis C virus (HCV)-related HCC. We identified this differentially expressed protein as complement C3a. The expression of C3a in HCC sera was further validated by PS20 chip immunoassay and Western blotting. Complement C3a was found to be elevated in patients with chronic hepatitis C and HCV-related HCC. The combination of SELDI-TOF MS and 2-DE provides a solution to identify disease-associated serum biomarkers.     

CK19 and Glypican 3 Expression Profiling in the Prognostic Indication for Patients with HCC after Surgical Resection

This retrospective study was designed to investigate the correlation between a novel immunosubtyping method for hepatocellular carcinoma (HCC) and biological behavior of tumor cells. A series of 346 patients, who received hepatectomy at two surgical centers from January 2007 to October 2010, were enrolled in this study. The expressions of cytokeratin 19 (CK19), glypican 3 (GPC3), and CD34 were detected by immunohistochemical staining. The clinical stage was assessed using the sixth edition tumor–node–metastasis (TNM) system (UICC/AJCC, 2010).Vascular invasion comprised both microscopic and macroscopic invasion. The tumor size, lymph node involvement, and metastasis were determined by pathological as well as imaging studies. Recurrence was defined as the appearance of new lesions with radiological features typical of HCC, seen by at least two imaging methods. Survival curves for the patients were plotted using the Kaplan–Meier method, and differences between the curves were assessed using the log-rank test. Significant differences in morphology, histological grading, and TNM staging were observed between groups. Based on the immunohistochemical staining, the enrolled cases were divided into CK19+/GPC3+, CK19−/GPC3+ and CK19−/GPC3− three subtypes. CK19+/GPC3+ HCC has the highest risk of multifocality, microvascular invasion, regional lymph node involvement, and distant metastasis, followed by CK19−/GPC3+ HCC, then CK19−/GPC3−HCC. CK19+/GPC3+ HCC has the shortest recurrence time compared to other immunophenotype HCCs. CK19 and GPC3 expression profiling is an independent prognostic indicator in patients with HCC, and a larger sample size is needed to further investigate the effect of this immunosubtyping model in stratifying the outcome of HCC patients.     

Proteomic analysis of autoantibodies in patients with hepatocellular carcinoma

To detect autoantibodies that could be diagnostic markers for hepatocellular carcinoma (HCC), we analyzed serum autoantibodies comprehensively that showed immunoreactivity to proteins in tumor tissue obtained from patients with HCC. Fifteen paired samples of HCC tissue and corresponding nontumorous liver tissue as well as five normal liver tissue samples were used in the study. A combination of proteomics and SEREX (serologic analysis of recombinant cDNA expression libraries) technique was used. Tissue proteins were separated by 2-DE, transferred onto PVDF membranes, and immunoblotted with autologous sera. By comparing each immunoblot pattern, we identified four immunoreactive spots with stronger staining intensity in tumorous tissues than in corresponding nontumorous tissues and in normal liver tissues. Matched proteins on 2-DE gels were identified by LC-MS/MS. These immunoreactive proteins were heat shock 70 kDa protein 1 (HSP70), glyceraldehyde 3-phosphate dehydrogenase, peroxiredoxin, and manganese superoxide dismutase (Mn-SOD). In HCC sera, occurrences of autoantibodies against these proteins were 7/15 (46.7%), 5/15 (33.3%), 5/15 (33.3%), and 6/15 (40.0%), respectively, whereas 2/20 (10.0%), 7/20 (35.0%), 0/20 (0.0%), and 2/20 (10.0%) were in control sera. Immunoblot analysis using commercially available purified proteins was performed to confirm the specificity of autoantibodies. By statistical analysis, autoantibodies against HSP70, peroxiredoxin, and Mn-SOD showed significantly high-frequency immunoreaction in HCC sera. The three antibodies were considered patient-specific antibodies in HCC and may be candidate diagnostic biomarkers for HCC.     

Heat-shock protein 27: a potential biomarker for hepatocellular carcinoma identified by serum proteome analysis

Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and ranks second in China. The prognosis of HCC remains dismal mainly because of its late diagnosis, especially in patients with coexisting chronic liver diseases. To identify serum biomarkers for HCC, sera from 20 healthy volunteers, 20 hepatitis B virus (HBV) infected patients and 20 HCC patients were selected for screening study and same number of sera into the same three groups were used for validation study. A strategy including sonication, albumin and immunoglobulin G (IgG) depletion and desalting was optimized for screening differentially expressed proteins of low abundance in serum. By 2-DE image analysis and MALDI-TOF-MS/MS identification, eight proteins including heat-shock protein 27 (HSP27), alpha-fetoprotein (AFP), alpha-1 antitrypsin, clusterin, caeruloplasmin, haptoglobin alpha2 chain, tranferrin and transthyretin were found significantly changed among the healthy, HBV and HCC groups. Further validation study by Western blot showed the detection of HSP27 in 90% HCC sera and two HBV sera, but in none of normal sera. Thus, 2-DE based serum proteome analysis can be useful in the screening of serum biomarkers for HCC and HSP27 could aid in the diagnosis of HCC though further validation is needed.