Carcinoma, Hepatocellular ; metabolism ; Glypicans ; biosynthesis ; genetics ; Humans ; Liver Neoplasms ; metabolism ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo investigate the correlation between the expressions of glypican-3 (GPC3) and Notch1 and their roles in the tumorigenesis and progression of hepatocellular carcinoma (HCC).

METHODSImmunohistochemistry and computerized image analysis were utilized to quantitatively detect the expressions of GPC3 and Notch1 in 30 HCC tissue specimens.

RESULTSIn the 30 HCC specimens, GPC3 expression decreased significantly as the grade of tumor differentiation increased (P<0.05 or P<0.01), while Notch1 expression presented with a reverse pattern of changes (P<0.05 or P<0.01). An obvious negative correlation was found between the expressions of GPC3 and Notch1 in HCC tissues (rp=-0.607, P=0.000; r=-0.692, P=0.000).

CONCLUSIONThe expressions of GPC3 and Notch1 show a negative correlation in HCC, suggesting a possible mechanism for mutual regulation between them to contribute to the tumorigenesis and progression of HCC.

Adult ; Aged ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Female ; Glypicans ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; Receptor, Notch1 ; metabolism

Objective: To clarify the diagnostic value of magnetic resonance imaging based on liver imaging reporting and data system (LI-RADS) for phosphatidylinositol proteoglycan-3 (GPC3) expression in hepatocellular carcinoma (HCC). Methods: Clinical and pathological data of 95 HCC cases with positive GPC3 expression (+) and 40 HCC cases with negative GPC3 expression (-) were retrospectively analyzed, and their MRI image features based on the 2018 version of LI-RADS were compared. Multivariate logistic regression analysis was used to determine the main predictors of GPC3 expression. Receiver operating characteristic curve was used further to determine the diagnostic efficacy of combined clinical imaging model to predict GPC3 expression. Enumeration data were compared with χ² test or Fisher's exact test. Measurement data were compared using independent samples t-test or Mann-Whitney U-test. Results: There were statistically significant differences between HCC in GPC3 (+) and GPC3(-) group at alpha-fetoprotein (AFP) levels (χ²=31.814, P<0.000 1), and MRI features: capsular enhacement (χ²=4.108, P=0.043), halo type enhancement (χ²=4.847, P=0.028), and lesion apparent dispersion coefficient (ADC) (t=2.552, P=0.011 8). Multivariate regression analysis showed that AFP>20 μg/L (OR=9.358, P<0.000 1) and ADC≤1.404×10^-3 mm²/s (OR=1.003, P=0.017) were independent predictors for GPC3 expression in HCC. The combined model and the area under the curve value for the diagnosis of GPC3(+) in HCC was 0.810, and its diagnostic sensitivity and specificity were 76.8% and 77.5%, respectively. Conclusion: AFP>20 μg/L and ADC≤1.404×10^-3 mm²/s may indicate the expression of GPC3 in HCC, and the combination of the two diagnostic indicators can provide a simple and effective non-invasive diagnostic method for clinical practice.
Biomarkers, Tumor ; Carcinoma, Hepatocellular/pathology* ; Glypicans/metabolism* ; Humans ; Liver Neoplasms/pathology* ; Magnetic Resonance Imaging ; Phosphatidylinositols ; Retrospective Studies ; alpha-Fetoproteins/metabolism*

OBJECTIVETo study the different expressions of glypican-3 in lung squamous cell carcinoma and adenocarcinoma and explore the association of glypican-3 with the prognosis of the patients.

METHODSGlypican-3 expression was detected immunohistochemically in the tumor tissues and adjacent tissues from 48 cases of lung squamous cell carcinoma and adenocarcinoma. Kaplan-Meier method and log-rank test were used for survival analysis of the patients.

RESULTSGlypican-3 expression was detected in the tumor tissues in 29.2% (14/48) of the cases, but not in the adjacent tissues. Of the 22 patients with lung squamous cell carcinoma, 12 (54.5%) showed positive glypican-3 expression in the tumor tissue, a rate significantly higher than that in patients with lung adenocarcinoma [7.7% (2/26), P<0.01]. In all the glypican-3-positive cases, the tumor tissues showed stronger glypican-3 expression in cases with lymph node metastasis or poor tumor differentiation. Kaplan-Meier survival analysis did not indicate a significant correlation of glypican-3 expression with the prognosis of the lung cancer patients.

CONCLUSIONPatients with lung squamous cell carcinoma have higher glypican-3 expressions in the tumor tissues than those with lung adenocarcinoma, suggesting the value of glypican-3 protein as a potential marker to detect lung squamous cell carcinoma.

Adenocarcinoma ; metabolism ; pathology ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; Glypicans ; metabolism ; Humans ; Kaplan-Meier Estimate ; Lung Neoplasms ; metabolism ; pathology ; Paraffin Embedding ; Prognosis

OBJECTIVETo explore the expression and diagnostic significance of glypican-3 (GPC3) in hepatoblastoma.

METHODSFive tissue microarray paraffin blocks were constructed to include 54 cases of hepatoblastoma. The tumor tissue samples were obtained from 3 surgical biopsies, 33 needle biopsies, 5 stage I resection tumors, and 13 stage II resection tumors after transcatheter arterial chemoembolization. Ten samples of non-neoplastic hepatic tissue adjacent to tumor were used as control. Immunohistochemical staining of GPC3 (clone 1G12) was performed. Among the 54 cases of hepatoblastoma, 22 cases were fetal subtype, 24 cases were mixed fetal and embryonal subtype and 8 cases were mixed epithelial and mesenchymal type.

RESULTSGPC3 was positive in fetal epithelial cells (54/54, 100%), but negative or weakly positive in embryonic epithelial cells in all cases of hepatoblastoma. Undifferentiated small cells and all mesenchymal components were negative for the expression. Non-neoplastic hepatocytes adjacent to tumor were negative for GPC3 expression (0/10) .

CONCLUSIONSFetal epithelial components of hepatoblastoma express GPC3 protein detectable by immunohistochemistry. Normal hepatocytes after birth, small cell undifferentiated and embryonic epithelial components of hepatoblastoma do not or weakly express GPC3 protein. Therefore, GPC3 immunohistochemistry offers a valuable aid to the diagnosis of hepatoblastoma in infants and children.

Child ; Child, Preschool ; Diagnosis, Differential ; Epithelial Cells ; metabolism ; Female ; Glypicans ; metabolism ; Hepatoblastoma ; diagnosis ; metabolism ; pathology ; Humans ; Immunohistochemistry ; Infant ; Infant, Newborn ; Liver Neoplasms ; diagnosis ; metabolism ; pathology ; Male

OBJECTIVETo study the expression and significance of GPC3, CD10 and CD34 in hepatocellular carcinoma (HCC), dysplastic nodules (DN), cirrhotic regenerative nodules (CRN), focal nodular hyperplasia (FNH) and hepatocellular adenoma (HA).

METHODSImmunohistochemical study for GPC3, CD10, CD34 and AFP was performed on 80 cases of HCC (30 cases of well-differentiated HCC and 50 cases of advanced HCC), 30 cases of DN (18 cases of high-grade DN and 12 cases of low-grade DN), 36 cases of CRN, 20 cases of FNH and 20 cases of HA.

RESULTS(1) The positive expression rate of GPC3 was 92% (46/50) in advanced HCC, 66.7% (20/30) in well-differentiated HCC, 2/18 in high-grade DN, and 0 in low-grade DN, CRN, FNH and HA. The expression rate of GPC3 in well-differentiated HCC was lower than that in advanced HCC and higher than that in high-grade DN (P < 0.05). (2) The negative expression rate of CD10 was 78% (39/50) in advanced HCC, 43.3% (13/30) in well-differentiated HCC, 20% (4/20 and 4/20) in both FNH and HA, 2.8% (1/36) in CRN and 0 in both high-grade DN and low-grade DN. The occurrence of CD10-strongly positive cells was 2% (1/50) in advanced HCC, 16.7% (5/30) in well-differentiated HCC, 15/18 in high-grade DN, 11/12 in low-grade DN, 80.6% (29/36) in CRN and 60% (12/20 and 12/20) in both FNH and HA. The positive expression rate of CD10 in well-differentiated HCC was higher than that in advanced HCC and lower than that in high-grade DN, low-grade DN, CRN, FNH and HA (P < 0.05). (3) The positive expression rates of CD34 in advanced HCC and well-differentiated HCC ranged from 25% to 100% [and strongly positive in 76% (38/50) and 70% (21/30), respectively]. The rates in high-grade DN and low-grade DN ranged from 5% to 25% (and weakly positive in 16/18 and 10/12, respectively). In CRN, the rate ranged from 0 to 5% [and weakly positive in 27.8% (10/36)]. In FNH and HA, the positive rates ranged from 25% to 50%. The positive expression rate of CD34 in well-differentiated HCC was significantly higher than that in high-grade DN, low-grade DN, CRN, FNH and HA (P < 0.05). (4) The positive expression rate of AFP was 44% (22/50) in advanced HCC, 20% (6/30) in well-differentiated HCC, no expression in DN, LCN, LCN, FNH and HA. The positive expression rate of AFP in well-differentiated HCC was lower than that in advanced HCC and higher than that in LCN, FNH and HA. The different expression had statistical significance (P < 0.05).

CONCLUSIONSGPC3 is a relatively sensitive and specific marker in pathologic diagnosis of HCC. When coupled with immunohistochemical results of CD34, CD10 and AFP, GPC3 is useful in differentiating HCC from DN, LCN, FNH and HA.

Adenoma, Liver Cell ; metabolism ; pathology ; Antigens, CD34 ; metabolism ; Biomarkers, Tumor ; metabolism ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Diagnosis, Differential ; Focal Nodular Hyperplasia ; metabolism ; pathology ; Glypicans ; metabolism ; Humans ; Immunohistochemistry ; Liver Cirrhosis ; metabolism ; pathology ; Liver Neoplasms ; metabolism ; pathology ; Neprilysin ; metabolism ; alpha-Fetoproteins ; metabolism

Adoptive immunotherapy based on chimeric antigen receptor-modified T cells (CAR-T) is one of the most promising strategies to treat malignant tumors, but its application in solid tumors is still limited. Glypican-3 (GPC3) is a meaningful diagnostic, therapeutic, and prognostic biomarker for hepatocellular carcinoma (HCC). The second/third generation GPC3-targeted CAR-T cells are generated to treat HCC. In order to improve the therapeutic effect, we constructed a fourth-generation lentiviral vector to express GPC3 CAR, human interleukin-7 (IL-7) and CCL19. Then the lentiviral vector and packaging plasmids were co-transfected into HEK293T cells to generate CAR lentiviral particles. Human T lymphocyte cells were transduced with CAR lentiviral to develop the fourth-generation GPC3-targeted CAR-T cells (GPC3-BBZ-7×19). In vitro, we used cell counting, transwell assay, luciferase bioluminescence assay and flow cytometry to compare the proliferation, chemotaxis, cytotoxicity and subtype distribution between GPC3-BBZ-7×19 CAR-T cells and the second generation GPC3-targeted CAR-T cells (GPC3-BBZ). In vivo, we established GPC3-positive HCC xenograft model in immunodeficient mice, then untransduced T cells (non-CAR-T) or GPC3-BBZ-7×19 CAR-T cells were injected. Tumor growth in mice was observed by bioluminescence imaging. Results showed that compared with GPC3-BBZ CAR-T, GPC3-BBZ-7×19 CAR-T cells had stronger proliferation, chemotactic ability, and higher composition of memory stem T cells (Tscm) (P values<0.05). However, there were no significant difference in cytotoxicity and cytokine secretion between them. In addition, GPC3-BBZ-7×19 CAR-T cells could significantly eliminate GPC3-positive HCC xenografts established in immunodeficient mice. Therefore, the fourth-generation GPC3-targeted CAR-T cells (secreting IL-7 and CCL19) are expected to be more durable and effective against HCC and produce tumor-specific memory, to provide a preclinical research basis for future clinical trials.
Animals ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Chemokine CCL19 ; metabolism ; Glypicans ; metabolism ; HEK293 Cells ; Humans ; Interleukin-7 ; metabolism ; Lentivirus ; genetics ; Liver Neoplasms ; Mice ; Receptors, Chimeric Antigen ; metabolism ; T-Lymphocytes ; metabolism ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the expression features of glypican-3 (GPC-3) and its diagnostic and differential values in hepatocellular carcinoma (HCC).

METHODSRat hepatoma models were made and the dynamic expression features of GPC-3 protein and its gene were investigated by Western blotting and RT-PCR respectively. Liver specimens from 36 HCC patients were collected by self-control method and the expression and clinicopathological features of GPC-3 were analyzed by immunohistochemistry. Serum GPC-3 levels were quantitatively detected by ELISA and its efficiency for HCC diagnosis was evaluated in patients with liver diseases.

RESULTSThe incidence of GPC-3 was 0% in control, 83.3% in degeneration, 100% in precancerosis and 100% in canceration during dynamic formation of rat hepatoma, respectively. The positive GPC-3 was brown granule- like staining localized in membrane and cytoplasm in human HCC.

CONCLUSIONSThe GPC-3 positive rates were 80.6% in HCC, 41.7% in surrounding tissues and none in distal tissues (P < 0.01), respectively. No positive relationship presented between GPC-3 and differentiation grade or the number of tumor except of tumor size (Z = 2.941, P < 0.01). The incidence of serum GPC-3 was 52.8% in HCC patients except of one patient with cirrhosis. No significant differences were found between GPC-3 and sex, age, AFP, tumor number, Child classification or extrahepatic metastasis except of tumor size (χ² = 6.318, P < 0.05) and HBV infection (χ² = 23.362, P < 0.01). Combined detection of GPC-3 and AFP could rise up diagnosis of HCC. GPC-3 expression closely associated with HCC and might be useful for early diagnosis of HCC.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Animals ; Carcinoma, Hepatocellular ; diagnosis ; metabolism ; pathology ; Diagnosis, Differential ; Female ; Glypicans ; metabolism ; Humans ; Liver ; pathology ; Liver Neoplasms ; diagnosis ; metabolism ; pathology ; Male ; Middle Aged ; Rats ; Rats, Sprague-Dawley ; Young Adult

OBJECTIVETo investigate the expressions of glypican-3 (GPC3) mRNA in hepatocellular carcinoma (HCC) tissues and peripheral blood cells (PBCs), and to determine the values of GPC3 mRNA in the diagnosis of HCC and HCC micrometastasis.

METHODSUsing semi-quantitative and nested reverse transcription polymerase chain reactions (RT-PCR), we detected the expressions of AFP and GPC3 genes in the tissues of 41 HCC, 41 paracancer and 52 non-HCC liver samples (41 far from HCC tissues and 11 normal liver tissues), and in the PBCs of 67 specimens from subjects.

RESULTSThe semi-quantitative RT-PCR displayed GPC3 mRNA was expressed in all samples of tissues and PBCs, and the relative intensities of its expressions in HCC, paracancer, non-HCC liver tissues were 78.9 +/- 35.5, 30.6 +/- 21.6, 23.8 +/- 15.5 respectively. The AFP mRNA expression values were 61.2 +/- 32.6, 31.5 +/- 23.6, and 21.2 +/- 15.9 respectively. The expression of each gene in HCC differed significantly from those in other two kinds of tissue samples (P < 0.01). The expressions of GPC3 mRNA and AFP mRNA, accounting for 80.5% and 63.4% in all the HCC tissues, were higher than their respective peak values in the tissues of non-HCC liver (+1.96s), but the expressions of at least one of the two genes was elevated in 92.7% of all the HCC tissues. There was a significant difference between combined detection of two genes and single AFP mRNA detection in HCC tissues (P < 0.01). Clinicopathologically, AFP mRNA was related with the grade of HCC and serum AFP, while GPC3 mRNA was related with not only the grade of HCC but also the invasion of HCC. The relative intensities of GPC3 mRNA expressions in PBCs of 67 specimens was 15.9 +/- 9.0, and GPC3 mRNA expressed in three kinds of tissue samples were all stronger than its counterparts in PBCs (P < 0.01). The GPC3 mRNA expression values in PBCs of the HCC group and the non-HCC group were respectively 16.1 +/- 8.3, 15.6 +/- 10.2, there was no significant difference between the two groups. Of the HCC metastasis group and the HCC non-metastasis group, the respective GPC3 mRNA expression values in PBCs were 16.0 +/- 9.0 and 16.3 +/- 7.7, there was also no significant difference between the two groups. The nested RT-PCR showed that the positive rates of AFP mRNA expressions in PBCs from the HCC group and the non-HCC group were 56.1% and 23.1%, and the difference between the two groups was significant (P = 0.011). The positive rates of AFP mRNA expressions in PBCs from the HCC metastasis group and the HCC non-metastasis group were 80.9% and 30.0%, and there was also a significant difference between the two groups (P = 0.002).

CONCLUSIONSAlthough GPC3 mRNA is expressed broadly, it still may serve as a potential tissue biomarker in the diagnosis of HCC. Detecting the expression of the two genes in the tissues will improve the screening and diagnosis of HCC. GPC3 is prevalently transcribed in the PBCs, but we have not found any relationship between the GPC3 expression in PBCs and the metastasis or recurrence of hepatocellular carcinoma, thus we can not identify HCC micrometastasis with GPC3 mRNA.

Adult ; Aged ; Carcinoma, Hepatocellular ; diagnosis ; metabolism ; pathology ; Female ; Glypicans ; biosynthesis ; genetics ; Humans ; Liver Neoplasms ; diagnosis ; metabolism ; pathology ; Male ; Middle Aged ; Neoplasm Metastasis ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; alpha-Fetoproteins ; biosynthesis ; genetics

OBJECTIVETo construct the expression vectors of GPC-3 CTL epitope.

METHODSThe HBsAg gene with three different EYILSLEEL (EYI) sites was named EYI1, and another with one EYI replacing CTL epitope FLG or SIL of pcHBsAg were named EYI2 and EYI3, respectively. All the three DNAs were amplified by SEOing PCR from pcHBsAg plasmid and linked into pBSSK+ vector to construct Pbssk/EYI1, pBSSK/EYI2, and pBSSK/EYI3. The three plasmid were identified by PCR, double digestion and sequencing, and the fragments with EYI1-3 were obtained by double digestion and then inserted into pcDNA3.1+ vector.

RESULTS AND CONCLUSIONPCR, enzyme digestion and sequence analysis confirmed successful construction of the eukaryotic expression vectors pcDNA-EYI1/HBsAg, pcDNA-EYI2/HBsAg, pcDNA-EYI3/HBsAg, which facilitate further studies of the GPC3-HBsAg multiple peptides vaccine for HBV infection.

Amino Acid Sequence ; Epitopes ; chemistry ; genetics ; immunology ; Genetic Vectors ; genetics ; Glypicans ; genetics ; immunology ; metabolism ; Hepatitis B Surface Antigens ; genetics ; immunology ; Plasmids ; genetics ; metabolism ; Polymerase Chain Reaction ; T-Lymphocytes, Cytotoxic ; immunology ; Vaccines, Subunit ; chemistry ; genetics ; immunology