The Basic Study For Clinical Value Of Tumor Biomarker-Glypican-3

Background/Purpose:Primary hepatocellular carcinoma(HCC) is one of the most common malignancies in the world.The incidence of HCC is rapidly increasing expecially in China.This is not only a medical issue but also a social problem.With the advancement of imaging analyses techniques,more and more hepatic nodular lesions could be detected.But we could not indentify the nature of pathological changes only by this methods,then to do pathological examination is great significant for diagnosis.However,so far there is no ideal of immunohistochemical markers can be used to identify the nature of liver nodular lesions.In recent years,more study showed that glypican-3(GPC3) protein which could be used as a marker of tumor structure were specificly over-expressed in HCC and hepatoblastoma.In addition GPC3 as a membrane glycoprotein,the soluble fragment(N-terminal) could be secreted and detected,so GPC3 protein may have improtant clinical value in serodiagnosis.After Komori H,et al.reported GPC3 protein contained two CTL epitopes in 2006,the focus of GPC3 research were not only on the diagnosis value but also on the immunotherapy value.The purpose of our job were to evaluate the clinical value of GPC3 protein in diagnosis and differential diagnosis and anti-tumor immunotherapy by detecting the expression of glypican-3(GPC3) in hepatocellular carcinoma tissues and GPC3 protein in the serum of HCC patients,and indentifing the immunological role of the two HLA-restricted CTL epitopes in GPC3 protein.Methods:The expression of GPC3 in 10 cases of poorly differentiated hepatocellular carcinoma,10 cases of adjacent tissues and 10 cases liver cirrhosis were detected by immunohistochemistry.We used the commodificated ELISA test kit to detect GPC3 protein in the serum of HCC,liver cirrhosis,chronic hepatitis,a variety of liver disease patients and gastrointestinal cancer patients and healthy people.In 45 HCC cases,we have detected both GPC3 protein and AFP and also detected them in the blood samples of five HCC patients before and after the propotional therapy,such as TAE,RF and/or photon knife.In additin,our study confirmed that GPC3 were specificly over-expression in HCC and wanted to discuss whether GPC3 can be used as a new target for anti-tumor immune.Two GPC3 CTL epitope peptides:A peptide(GPC3_298-306,EYILSLEEL) and B peptide(GPC3_144-152, FVGEFFTDV),expressed in vitro and partly be coupled with KLH were used as immunogens to induced immunity in BALB/c mice.Divided BALB/c mice into six groups in random,4 experimental groups and 2 control groups,each group contained six mice.The mice in experimental group 1 and 2 were immunized A and B peptide respectively;The mice in experimental group 3 and 4 were immuned A-KLH and B-KLH respectively;Group 5 and 6 were control groups primed with KLH and saline. 7 days after the last immunization,mouse spleen were seperated to isolate lymphocytes,added the corresponding stimulus,and then countered IFN-γ,spots secreted by actived T cells through ELISPOT to determine whether the two peptides could induce specific immune responses.Observed the deferences in percentege of CD4⁺ T cell and CD8⁺ T cell by flow cytometry in order to judge the immune response in mice,particularly the cellular immune.Results:The brown particles evenly distributed in hepatoma cell membrane and the cytoplasm were considered as positive performance.The immunohistochemistry results showed that GPC3(+) in seven cases of 10 HCC tissues epression(70%),two cases of that with low expression.While GPC3 expression were negative in all 10 cases of HCC adjacent tissues and 10 cases of liver cirrhosis.Moreover GPC3 expression was cell-specific,tumor tissue in the bile duct epithelial cells,fibroblasts, vascular endothelial cells and infiltrating lymphocytes were negative.ELISA showed that GPC3 protein in serum of HCC patients were significantly higher than those in other patients.GPC3 protein in 29 cases of 45 HCC patients was positive (29/45,64.4%);liver cirrhosis(2/35,5.7%);metastatic liver cancer(2/20,10%);liver cyst(1/15,6.7%).In the serum of 35 patients with chronic hepatitis,adiposis hepatica, and other liver diseases,gastric and colorectal cancer,and healthy people,GPC3 protein were negative.In 45 cases of HCC,AFP positive rate was 55.6%,while GPC3 positive diagnosis rate was 64.4%.AFP was negtive in 11 patients among the 29 GPC3 positive patients,and while GPC3 was negive in 7 patients among the 25 AFP positive patients.So we concluded that there were no relationship between AFP and GPC3 in the serum of HCC patients.Joint detection of GPC3 and AFP could be increase diagnosis rate to 80%.three cases of Ib tumors were found in GPC3 positive and AFP negative cases.After analysing 45 cases of HCC,we found that increased concentration of GPC3 protein in the patients serum were directly related with HCC, but not with liver cell injury caused by hepatitis and other benign liver diseases.In addition,we detected the blood samples of five HCC patients before and after the propotional therapy,such as TAE,RF and/or photon knife.The quantification of GPC3 protein in the serum decreased in accordence with the tumor size and number reductions,which showed a more stable than AFP.The data showed that the specificity and sensitivity of GPC3 were more better than AFP.ELISPOT results showed that in lymphocytes stimulated secretion of IFN-γ,levels,group 1,2 and group 6 were statistically significant differences(P＜0.05).The differences between Group 3,4 and all control groups were statistical significance(P＜0.05),while we could not find any significant differences among experimental groups(P＞0.05).The experiments were repeated twice with the same results.Flow cytometry showed the CD4⁺T/CD8⁺T was obviously increased after immunizing with the special peptide in experimental groups.The difference between experemental groups and control groups were significant(P＜0.05).Conclusions:GPC3 protein were overexpressed in HCC tissues,so immunohistochemical detection of GPC3 can be valuable as a mean for diagnosis and it may improve pathologic diagnosis of hepatocellular carcinoma.GPC3 and AFP used in the detection of the complementary role,were expected to improve the positive rate and specificity of early diagnosis of HCC and could be useful for analysing curative effect and prognosis.We proved two CTL epitope peptide GPC3_298-306 and GPC3_144-152 could be recognized by specific T cells through vivo experiment and induced activation of T cell,which triggered the specific immune response,especially the cellular immune.Our research provided a strong theoretical basis for anti-tumor immunotherapy targeted with GPC3.We could conclude that GPC3 protein had the more better clinical value.However,more deeper researches are urgently needed to prove whether GPC3 specific tumor immunotherapy can be widely used in clinical.