Glypican-3 mRNA Expression Research Articles

Glypican-3 Differentiates Intraductal Carcinoma and Paget's Disease from Other Types of Breast Cancer.

Background and Objectives: breast cancer remains the most common health burden affecting females worldwide. Despite developments in breast cancer diagnostic approaches and treatment strategies, the clinical management of metastatic breast cancer remains challenging. Thus, there is a need to identify new biomarkers and novel drug targets for breast cancer diagnosis and therapy. Recently, aberrant glypican-3 (GPC3) expression in cancers has gained considerable interest in cancer research. The studies, however, have yielded contradictory results about GPC3 expression in breast cancer. Therefore, the current study aims to analyse GPC3 expression across a large panel of different breast cancer subtypes. Materials and Methods: GPC3 expression was immunohistochemically evaluated in 230 breast cancer patients along with eight normal tissues and its associations to clinical and demographic characteristics, as well as immunohistochemical biomarkers for breast cancer. Moreover, a public database consisting of breast cancer patients' survival data and GPC3 gene expression information was used to assess the prognostic value of GPC3 in the survival of breast cancer patients. Results: GPC3 expression was only characterised in 7.5% of different histological breast cancer subtypes. None of the normal breast tissues displayed GPC3 expression. Interestingly, all cases of Paget's disease, as well as 42.9% of intraductal and 16.7% of mucinous carcinomas were found to have GPC3 expression, where it was able to significantly discriminate Paget's disease and intraductal carcinoma from other breast cancer subtypes. Importantly, GPC3 expression was found more often in tumours that tested positive for the expression of hormone receptors and human epidermal growth factor receptor 2 (HER2), indicating more favourable histological subtypes of breast cancer. Consequently, longer relapse-free survival (RFS) was significantly correlated with higher GPC3 mRNA expression. Conclusions: Our study proposes that GPC3 is a promising breast cancer subtype-specific biomarker. Moreover, GPC3 may have the potential to be a molecular target for the development of new therapeutics for specific subtypes of breast cancer.

Abstract 5415: Glypican-3 immunohistochemistry precision, validation, and prevalence in selected solid tumors to identify target populations for CAR T cell therapy

Abstract Background: Glypican-3 (GPC3) is an oncofetal tumor antigen that is an attractive target for chimeric antigen receptor (CAR) T cell therapy due to its highly restricted expression on normal tissue and high prevalence in several adult and pediatric solid tumors. Aberrant GPC3 expression is implicated in tumorigenesis, and GPC3+ cancers are characterized by a highly immunosuppressive landscape which induces exhaustion in tumor-resident T cells. We sought to assess the validity of a GPC3 antibody clone in immunohistochemistry (IHC) and then measure GPC3+ prevalence in selected solid tumors. Methods: We evaluated GPC3 transcripts in human normal and tumor tissue specimens from The Cancer Genome Atlas (TCGA) and the Genome-Tissue Expression Project. GPC3 IHC was performed on formalin fixed paraffin embedded tumor tissues: hepatocellular (HCC), squamous cell lung (SCC), colorectal (CRC), serous ovarian (OC), Merkel cell carcinomas (MCC) and liposarcoma (LS) using antibody clone GC33 and scored using H-score (range 0-300), with appropriate positive and negative controls. Accuracy and precision of GPC3 antibody clone GC33 were tested in GPC3+ high prevalence tumor types compared to a validated anti-GPC3 clone (1G12). GPC3+ cutoff was set as H-score ≥ 30 (GC33) compared to >20 (1G12). The prevalence of GPC3 expression measured by IHC was also used to calculate the theoretical mRNA cutoff value from the TCGA data for GPC3+ indications represented in the database. Results: GPC3 mRNA was expressed at low levels predominantly in lung, adipose, tibial nerve, kidney and breast. GPC3 IHC of normal tissue showed predominantly faint, cytoplasmic staining in a few tissues including heart, kidney and stomach and no expression in lung or breast. HCC, SCC, LS, CRC, and OC showed high and varying levels of GPC3 mRNA expression in TCGA samples. The theoretical mRNA expression cutoff value based on IHC were HCC=1396 RSEM, SCC= 2675 RSEM, Sarcoma= 4126 RSEM; and OC 4486 RSEM; respectively. A cutoff for CRC could not be determined because the prevalence of GPC3 by IHC was <10%. GPC3 IHC using GC33 showed a higher prevalence of positive cases in myxoid/round cell LS (MRCLS) relative to other LS subtypes (15/45 vs. 8/75). HCC (n=110), SCC (n=73), MRCLS (n=45), and MCC (n=20) had a prevalence of 69%, 33%, 33%, and 70%; respectively. However, <10% of CRC (n=80) or OC (n=31) were GPC3+. GPC3 IHC assay using clone GC33 was fully validated for use as a lab-developed test by assessing accuracy, sensitivity, specificity, and precision in SCC, HCC, MCC and LS specimens. Conclusions: GPC3 has a high prevalence in selected tumors with low level expression in some normal tissues, making it an attractive target for CAR T cell therapy. The GC33 clone performs similarly to 1G12 and could be used for IHC screening for CAR T cell therapy. MRCLS has higher prevalence of GPC3+ expression relative to other LS subtypes. Citation Format: Glen J. Weiss, Amy Jensen-Smith, Sujatha Muralidharan. Glypican-3 immunohistochemistry precision, validation, and prevalence in selected solid tumors to identify target populations for CAR T cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5415.

Cobalt Chloride Induced Apoptosis by Inhibiting GPC3 Expression via the HIF-1α/c-Myc Axis in HepG2 Cells.

PurposeTo investigate the role of glypican-3 (GPC3) in cobalt chloride (CoCl2)-induced cell apoptosis in hepatocellular carcinoma.MethodsHepG2 cells were treated with CoCl2 in the absence or presence of GPC3 plasmid transfection. Cell viability and apoptosis were assessed by MTT assay and flow cytometry, respectively. The expression of GPC3, hypoxia-inducible factor 1α (HIF-1α), c-myc, sp1, poly-ADP-ribose polymerase (PARP) and caspase-3 was determined by real-time PCR, Western blotting, and immunofluorescence after the cells were treated with different concentrations of CoCl2 or siRNA targeting HIF-1α.ResultsCoCl2 significantly inhibited the proliferation of HepG2 cells and induced apoptosis. Additionally, the expression of GPC3 mRNA and protein was decreased, and overexpression of GPC3 attenuated the tumour inhibiting effects. Further studies showed that CoCl2 increased the expression of HIF-1α while reducing the expression of sp1 and c-myc; knockdown of HIF-1α elevated the expression of GPC3, sp1, and c-myc.ConclusionCoCl2 inhibited the growth of HepG2 cells through downregulation of GPC3 expression via the HIF-1α/c-myc axis.

Hepatic glypican-3 and alpha-smooth muscle actin overexpressions reflect severity of liver fibrosis and predict outcome after successful portoenterostomy in biliary atresia

BackgroundGlypican-3 plays a vital role in regulating embryonic morphogenesis of the liver. This study aimed to investigate associations of hepatic expressions of glypican-3 and alpha-smooth muscle actin with clinical parameters in biliary atresia. MethodsLiver specimens were obtained from 20 biliary atresia infants and 7 non-biliary atresia controls. Relative mRNA expressions of glypican-3, alpha-smooth muscle actin, and signaling molecules of Wnt/β-catenin were measured using real-time polymerase chain reaction. Protein expressions of glypican-3 and alpha-smooth muscle actin were examined using immunohistochemistry. Masson’s trichrome staining was conducted to evaluate the stage of liver fibrosis. ResultsUp-regulation of glypican-3 mRNA expression was observed in biliary atresia livers, and its expression was positively associated with alpha-smooth muscle actin, β-catenin, c-Myc, and cyclin D-1. Immunostaining scores of glypican-3 and alpha-smooth muscle actin were significantly increased in biliary atresia livers. Biliary atresia patients with poor outcomes had significantly greater glypican-3 expression than those with good outcomes, consistent with hepatic alpha-smooth muscle actin expression analysis. Hepatic glypican-3 expression was associated with age, albumin, aspartate transaminase, and alkaline phosphatase in biliary atresia patients, while hepatic alpha-smooth muscle actin expression was correlated with alkaline phosphatase in the patients. Moreover, glypican-3 and alpha-smooth muscle actin expressions were positively associated with fibrosis stage in biliary atresia livers. There was a positive relationship between glypican-3 and alpha-smooth muscle actin expression in biliary atresia livers. Combined high expressions of glypican-3 and alpha-smooth muscle actin were associated with poor survival. ConclusionHepatic overexpressions of glypican-3 and alpha-smooth muscle actin were associated with hepatic dysfunction and the degree of liver fibrosis in biliary atresia.

Glypican-3 expression in malignant small round cell tumors.

Malignant small round cell tumors usually progress rapidly and show resistance to chemotherapy, and it is often difficult to make a definitive diagnosis based on their histological morphology. Glypican-3 (GPC3) is a highly tumor-specific antigen, and the overexpression of GPC3 was reported in many pediatric and adult malignancies. In the present study, we investigated the GPC3 expression in pediatric malignant small round cell tumors to assess its role in the differential diagnosis of the tumors. Immunohistochemistry was performed to assess the expression of GPC3 in samples from 84 rhabdomyosarcomas (RMSs; 44 alveolar and 40 embryonal RMSs), 62 Ewing sarcomas (EWSs), 35 neuroblastomas (NBs) and two desmoplastic small round cell tumors (DSRCTs). We performed a reverse transcription-quantitative polymerase chain reaction for GPC3 to determine the GPC3 mRNA expression in samples from 66 frozen tumors (23 RMSs, 28 EWSs and 15 NBs). The serum expression levels of GPC3 were analyzed in pre-operative blood samples from two RMS and eight NB patients. In total, 25% (21/84) of the RMSs and 3% (1/35) of the NBs exhibited a focal expression of GPC3, whereas, the other specimens showed no GPC3 expression. The GPC3 mRNA expression level of the RMSs with positive GPC3 expression (n=6) was significantly higher compared with the RMSs without such expression (n=17). A total of two cases of NB showed high serum levels of GPC3, but neither tumor showed immunoreactivity for GPC3. The immunohistochemical overexpression of GPC3 may be a candidate ancillary parameter in the differential diagnosis of RMS from EWS and DSRCT.

Glypican-3 mRNA expression level in Wilms tumor: correlation with histological type, stage, and outcome.

To correlate expression of Glypican-3 in Wilms tumor with histopathology, stage, and outcome. Glypican-3 mRNA expression by real-time PCR on tumor and normal germline samples from 75 fresh nephrectomies for Wilms tumor with fold change after normalization against GAPDH was compared. Survival analysis for event-free and overall survival (EFS, OS) with 2-year follow-up for Glypican-3 overexpression (>1.5 times) and clinicopathological parameters was performed. Glypican-3 was overexpressed in 37/75 (49.3%). It was overexpressed in 77% (10/13) cases with blastema predominance or anaplastic histology, as compared to 44% of other histologies (27/62) (p=0.03). OS was 73 and 93%, respectively (p=0.016), for those with and without GPC-3 overexpression. EFS was not significantly different with Glypican-3 overexpression (p=0.11). All 5 deaths among blastema predominant tumors and 4/5 deaths among triphasic tumors had overexpressed Glypican-3. Most deaths in Stage IV, Stage III, and Stage I+II (5/7, 3/3, 1/1) had GPC-3 overexpression. On multivariate analysis, only histology and stage were found to have independent prognostic value. Glypican-3 overexpression in Wilms tumor correlates with poor OS on univariate analysis. However, only histology and stage have independent prognostic value. Glypican-3 levels may help to stratify intermediate outcome histology (triphasic) and Stage III Wilms tumors.

The Value of GPC3 and GP73 in Clinical Diagnosis of Hepatocellular Carcinoma.

The incidence of hepatocellular carcinoma (HCC) has increased over the past decades in China. Current screening methods of HCC such as detection of α-fetoprotein (AFP) combined with liver ultrasonography remain unsatisfactory. Many HCC patients have already missed the optimal treatment period when diagnosed. Our study aimed to evaluate the value of Glypican 3 (GPC3) and Golgi protein 73 (GP73) in the detection of HCC. Thirty-nine patients with HCC and 31 patients with liver cirrhosis were enrolled. The level of serum GPC3 and GP73 were determined by ELISA. The expression of GPC3 mRNA and GP73 mRNA in peripheral blood mononuclear cell (PBMC) and liver tissues were also measured with qRT-PCR. Then, receiving operating characteristic (ROC) curves were plotted to detect the sensitivity and specificity of serum GPC3 and GP73 in the diagnosis of HCC. The levels of serum GPC3 and GP73 in the HCC group were significantly higher than in the cirrhosis group (p < 0.0001). Patients with GPC3 > 9.3 μg/L and GP73 > 77.68 ng/mL had a risk of HCC of 92.31%. The HCC diagnosis ROC curve analysis indicated that when setting the GPC3 cutoff value > 9.3 μg/L, AUC = 0.956. The sensitivity and specificity of GPC3 were 89.74% and 96.77%, respectively, with a positive predictive value of 97.2%, negative predictive value of 88.2%, + LR of 27.82 and - LR of 0.11. When setting GP73 cutoff value > 77.68 ng/mL, AUC = 0.937. The sensitivity and specificity of GP73 were 92.31% and 83.87%, respectively, with positive predictive value of 87.8%, negative predictive value of 89.7%, + LR of 5.72 and - LR of 0.092. No significant difference (p > 0.05) was found between GPC3 and GP73 AUC in ROC curves, indicating that these two biomarkers were equivalent in the prediction of HCC. The expression of serum GPC3 and GP73 was significantly higher in the HCC patients compared with the cirrhosis patients. GPC3 and GP73 might be effective non-invasive diagnostic indicators of HCC.

Differential Expression of Glypican-3 and Insulin–Like Growth Factor-II mRNAs and Alpha-Fetoprotein and Ki-67 Markers in HCV Related Hepatocellular Carcinomas In Egyptian Patients

Background:Increasing evidence indicates that in hepatocellular carcinomas (HCCs) abnormal gene expression, for example of glypican-3 (GPC-3) and insulin-like growth factor-II (IGF-II), are associated with the occurrence and progression of HCC. The objective of this study was to evaluate the differential expression of GPC-3 and IGF-II mRNAs in HCC tissues with a background of chronic hepatitis C virus (HCV) genotype 4 cirrhosis, in relation to Ki-67 and alpha-feto protein (AFP) tissue markers.Methods:One hundred and five patients with HCCs who had undergone hepatectomy, were included, after obtaining informed consent. Total RNA was extracted from malignant and corresponding peri-malignant liver tissues, and GPC-3 and IGF-II mRNAs in addition to beta-actin mRNA as an internal control, were evaluated in all samples by reverse transcriptase-polymerase chain reactions (RT-PCR). Routine histopathological diagnosis as well as immunohistochemical (IHC) staining using monoclonal antibodies for Ki-67 and AFP were also performed.Result:Expression of GPC-3 mRNA was positive in all HCC malignant tissue, with overexpression in 86/105 (81.9%); in respect to the grade of the tumor (1-3 grades), while in peri-malignant tissue it was over expressed only in 20/105 (19%). The IGF-II mRNA was over expressed in only 10/105 (9.5%) malignant and peri-malignant samples. AFP was expressed in 33.3% of malignant samples but absent in peri-malignant tissues. Ki-67 expression was significantly increased in malignant compared to peri-malignant tissue.Conclusion:GPC-3 and IGF II mRNAs may be good molecular markers for HCC, especially with a background of cirrhosis due to chronic HCV infection. Significant correlations were noted with the pattern of AFP and Ki-67 expression.

Expression of Glypican-3 (GPC3) in Malignant and Non-malignant Human Breast Tissues

Specific reports have linked GPC3 with cancer. Its usefulness as a marker has been proved for hepatocarcinoma, melanoma and ovary carcinoma. However, there are no studies analyzing GPC3 usefulness as a biomarker in mammary tumors. The aim of this work was to analyze GPC3 expression in breast tissues and to determine whether it might be useful as a biomarker in breast cancer patients. Expression level of GPC3 mRNA in Brazilian and Argentine human breast tumor (n=121) and peritumoral tissue (n=77) samples was analyzed using qRT-PCR. GPC3 protein expression was analyzed from 69 breast cancer and 10 peritumoral samples using IHC. Statistical analyses were done to evaluate the clinical-pathological significance of GPC3 expression. We found that Brazilian and Argentine populations are statistically different regarding GPC3 mRNA expression. In Argentine patients a lower GPC3 mRNA expression was found in tumors as compared to peritumoral tissues. No association was found between GPC3 mRNA and protein expression and the clinical-pathological parameters. The Kaplan-Meier curves suggested that elevated levels of GPC3 mRNA are associated with relapse. Our results indicate differential expression of GPC3 in mammary tumors in comparasion to normal breast tissues. They also suggest the potential role of GPC3 as a biomarker and the importance of deepening the study.

Establishment and biological characterization of a novel cell line derived from hepatoid adenocarcinoma originated at the ampulla of Vater

Hepatoid adenocarcinoma is a rare gastrointestinal tumor and mostly reported in the stomach. Effective chemotherapy has yet to be developed to improve poor prognosis. The present study was undertaken to establish a useful cell line derived from a hepatoid adenocarcinoma, possibly leading to a new therapeutic strategy. The new human cell line VAT-39 was established from a metastatic lymph node of a 69-year-old Japanese male patient with hepatoid adenocarcinoma of the ampulla of Vater. The primary tumor and metastatic lymph node were composed of hepatoid adenocarcinoma cells exhibiting immunohistochemical reactivity for alpha-fetoprotein (AFP) and glypican-3 (GPC3). In the metastatic lymph node, Periodic acid-Schiff (PAS) staining clarified diffuse deposition of glycogen in the cytoplasm, indicating analogous characteristics to the primary hepatoid adenocarcinoma. Moreover, VAT-39 cells produced high levels of AFP in the cultured medium, and reverse-transcriptase polymerase chain reaction (RT-PCR) verified increased expression of GPC3 mRNA in this cell line. Further, we evaluated the sensitivity to major chemotherapeutic drugs against the bile duct cancer. Neither 5-fluorouracil nor gemcitabine showed particular sensitivity to this cell line. The tumorigenicity of the cultured cells was confirmed in athymic nude mice and the histological features of the explanted tumor were similar to the VAT-39 cell line. The present VAT-39 is the first hepatoid adenocarcinoma cell line that originates from the ampulla of Vater and it will be applicable for basic biological studies searching for new strategies of molecular targeted chemotherapy to this disease.

Expression of Glypican-3 in Mouse Embryo Stem Cells and its Derived Hepatic Lineage Cells Treated with Diethylnitrosamine in vitro

To clarify the role of stem cells in hepatocarcinogenesis, glypican-3 (GPC-3) and E-cadherin expression was investigated in embryonic cell lineages. Mouse embryonic stem cells (ESCs), hepatic progenitor cells (HPCs) and hepatocyte like cells (HCs), representing 0, 22 and 40 days of differentiation, respectively, were treated in vitro with diethylnitrosamine (DEN) at four doses (0, 1, 5 and 15 mM; G1, G2, G3 and G4, respectively) for 24 h and GPC- 3 and E-cadherin expression was examined by relative quantitative real-time PCR and immunocytochemistry. GPC-3 mRNA expression was significantly different for G4 at day 0 (p<0.001) and for G4 at day 22 (p<0.01) compared with the control (G1). E-cadherin mRNA expression was significantly different for G3 and G4 at day 0 (p<0.05 and p<0.001, respectively), for G2 and G4 (p<0.05 and p<0.001, respectively) at day 22 and for G2 and G4 (p<0.01 and p<0.001, respectively) at day 40 compared with G1. Immunofluorescence staining for GPC-3 showed a membranous and/or granular expression in cytoplasm of ESCs and HPCs and granular and/or diffuse expression in cytoplasm of HCs, which were also stained by E-cadherin. DEN treatment increased GPC- 3 expression in ESCs, HPCs and HCs, with increase of E-cadherin expression. Taken together, the expression of GPC-3 was altered by DEN treatment. However, its expression pattern was different at the stage of embryo stem cells and its derived hepatic lineage cells. This suggests that GPC-3 expression may be modulated in the progeny of stem cells during their differentiation toward hepatocytes, associated with E-cadherin expression.

A functional screening identifies five micrornas controlling glypican-3: role of mir-1271 down-regulation in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the major primary liver cancer. Glypican-3 (GPC3), one of the most abnormally expressed genes in HCC, participates in liver carcinogenesis. Based on data showing that GPC3 expression is posttranscriptionally altered in HCC cells compared to primary hepatocytes, we investigated the implication of microRNAs (miRNAs) in GPC3 overexpression and HCC. To identify GPC3-regulating miRNAs, we developed a dual-fluorescence FunREG (functional, integrated, and quantitative method to measure posttranscriptional regulations) system that allowed us to screen a library of 876 individual miRNAs. Expression of candidate miRNAs and that of GPC3 messenger RNA (mRNA) was measured in 21 nontumoral liver and 112 HCC samples. We then characterized the phenotypic consequences of modulating expression of one candidate miRNA in HuH7 cells and deciphered the molecular mechanism by which this miRNA controls the posttranscriptional regulation of GPC3. We identified five miRNAs targeting GPC3 3'-untranslated region (UTR) and regulating its expression about the 876 tested. Whereas miR-96 and its paralog miR-1271 repressed GPC3 expression, miR-129-1-3p, miR-1291, and miR-1303 had an inducible effect. We report that miR-1271 expression is down-regulated in HCC tumor samples and inversely correlates with GPC3 mRNA expression in a particular subgroup of HCC. We also report that miR-1271 inhibits the growth of HCC cells in a GPC3-dependent manner and induces cell death. Using a functional screen, we found that miR-96, miR-129-1-3p, miR-1271, miR-1291, and miR-1303 differentially control GPC3 expression in HCC cells. In a subgroup of HCC, the up-regulation of GPC3 was associated with a concomitant down-regulation of its repressor miR-1271. Therefore, we propose that GPC3 overexpression and its associated oncogenic effects are linked to the down-regulation of miR-1271 in HCC.

Targeted glypican-3 gene transcription inhibited the proliferation of human hepatoma cells by specific short hairpin RNA

Hepatocellular carcinoma (HCC) is a highly chemoresistant cancer with no effective systemic therapy. Despite of surgical or locoregional therapies, prognosis remains poor because of high tumor recurrence or progression, and currently, there are no well-established effective adjuvant therapies. Glypican-3 (GPC-3) is specifically overexpressed in hepatoma and perhaps is a valuable molecular target for HCC therapy. In this present study, the effect of silencing GPC-3 gene transcription on human HepG2 cell proliferation was investigated by constructing GPC-3 short hairpin RNA (shRNA) plasmid. After HepG2 cells were transfected with the most efficient shRNA, GPC-3 mRNA expression (90.4 %) was inhibited significantly and estimated by fluorescence quantitative reverse transcriptase-polymerase chain reaction, and the result was accordance with downregulation at the protein level. The percentage of the cell proliferation was down to 28.9 % in the shRNA group and 19.9 % in the shRNA plus sorafenib group. The cell cycles were arrested in the G1 phase (65.6 %) and the apoptosis rate was increasing (66.75 %) in the shRNA1 group with significant alteration compared with that in the negative-shRNA group. Specific shRNA might intervene effectively GPC-3 activation and inhibit tumor cell proliferation, suggesting that GPC-3 gene should be a potential molecular target for HCC therapy.

Glypican-3 expression and its relationship with recurrence of HCC after liver transplantation

To investigate the diagnostic value of glypican-3 (GPC3) and its relationship with hepatocellular carcinoma (HCC) recurrence after liver transplantation. HCC tissue samples (n = 31) obtained from patients who had undergone liver transplantation were analyzed. GPC3 mRNA and protein expression were analyzed by TaqMan real-time reverse transcription-polymerase chain reaction and immunohistochemistry. Correlation between the GPC3 expression and clinicopathological features was analyzed. The potential prognostic value of GPC3 was investigated by comparing recurrence-free survival between HCC patients with and without GPC3 expression. Using a cutoff value of 3.5 × 10⁻², 20 of 31 cancerous tissues had expression values of > 3.5 × 10⁻², whereas 3 of 31 adjacent non-neoplastic parenchyma and 0 of 20 control liver tissues had expression values of > 3.5 × 10⁻² (P < 0.001). GPC3 protein was immunoexpressed in 68% of cancerous tissues, but not in adjacent non-neoplastic parenchyma and control liver tissues. Vascular invasion was significantly related to GPC3 expression (P < 0.05). Recurrence-free survival was significantly longer for patients without GPC3 mRNA overexpression (> 3.5 × 10⁻²) and those without vascular invasion (P < 0.05 for both). GPC3 expression may serve as a valuable diagnostic marker for HCC. GPC3 mRNA overexpression may be an adverse indicator for HCC patients after liver transplantation.

Oncofetal antigen glypican-3 as a promising early diagnostic marker for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is characterized by a multi-cause, multi-stage and multi-focus process of tumor progression. Its prognosis is poor and early diagnosis is of utmost importance. This study was undertaken to investigate the dynamic expression of oncofetal antigen glypican-3 (GPC-3) and GPC-3 mRNA in hepatocarcinogenesis and to explore their early diagnostic value for HCC. A hepatoma model was induced in male Sprague-Dawley rats with 0.05% 2-fluorenylacetamide and confirmed by hematoxylin and eosin staining and gamma-glutamyltransferase (GGT) expression. Total RNA was purified and transcribed into cDNA by reverse transcription. Fragments of the GPC-3 gene were amplified by nested RT-PCR, and confirmed by sequencing. GPC-3 was analyzed by immunohistochemistry, Western blotting or ELISA. Positive GPC-3 expression showed as brown granule-like staining localized in the cytoplasm. Histological examination of hepatocytes revealed three morphological stages of granule-like degeneration, atypical hyperplasia (precancerous), and cancer formation, with a progressive increase of liver total RNA and GGT expression. The incidence of liver GPC-3 mRNA and GPC-3, and serum GPC-3 was 100%, 100% and 77.8% in the HCC group, 100%, 100%, and 66.7% in the precancerous group, 83.3%, 83.3%, and 38.9% in the degeneration group, and no expression in the liver or blood of the control group, respectively. There was a positive correlation between liver GPC-3 mRNA and total RNA level (r=0.475, P<0.05) or liver GPC-3 (r=1.0, P<0.001) or serum GPC-3 (r=0.994, P<0.001). Abnormal oncofetal antigen GPC-3 and GPC-3 mRNA expression in hepatocarcinogenesis may be promising molecular markers for early diagnosis of HCC.

Glypican-3 is overexpressed in lung squamous cell carcinoma, but not in adenocarcinoma

Glypican-3 is a membrane-bound proteoglycan whose expression has been linked to malignancies through the existence of both mutations and aberrant protein expression. Reports on glypican-3 expression in lung cancer were limited, with some evidence for loss of expression, which suggested a tumor-suppressor role. We sought to evaluate glypican-3 expression in lung cancer at the protein and mRNA levels and correlate it with clinical, histological and genomic characteristics such as RAS mutation status. We used immunohistochemistry on tissue microarray to study glypican-3 expression in 97 patients, evaluated glypican-3 mRNA levels by quantitative polymerase chain reaction in 143 patients and identified RAS mutations by allele-specific oligonucleotide hybridization. We correlated the results with clinical and histological data. Glypican-3 immunostaining was negative in all normal lung tissues, but positive in 23% of lung carcinoma samples. High protein and mRNA expression was associated with squamous histology (positive stain in 55% of squamous cell carcinoma vs 8% of adenocarcinoma, P<0.0001 for both immunostaining and mRNA). RAS mutations were highly associated with adenocarcinoma and low glypican-3 mRNA expression (P<0.0001 for both). Among smokers, glypican-3 mRNA expression was reduced in adenocarcinoma patients (P=0.013), and was elevated in those with squamous cell carcinoma (P=0.03, interaction P=0.0009). These opposing associations also correlated with the smoking burden. Patients with tumors staining positively for glypican-3 smoked significantly more than patients with tumors staining negatively (P=0.013). No association was found between glypican-3 expression and patient outcome. In conclusion, glypican-3 was overexpressed in cancerous compared with normal lung tissue. Adenocarcinoma and squamous cell carcinoma had differential expression of glypican-3, with predilection to squamous cell carcinoma patients who smoked. Glypican-3 expression in squamous cell carcinoma as an oncofetal protein renders it a potential candidate marker for early detection of lung squamous cell carcinoma.

Immunohistochemical Study of Glypican 3 in Thyroid Cancer

In 123 patients with thyroid cancer, expression of glypican 3 (GPC3) was immunohistochemically investigated in tissue samples and the biological significance of GPC3 in thyroid cancer was examined. GPC3 was scarcely expressed in the normal thyroid gland, but was dramatically enhanced in certain types of cancers: 100% in follicular carcinoma (20/20 cases) and 70% in papillary carcinoma (48/69 cases). Expression of GPC3 in follicular carcinoma was significantly higher than that of follicular adenoma (p < 0.0019). In contrast, GPC 3 was not expressed in 17 cases of anaplastic carcinoma. A high expression of GPC3 mRNA was confirmed in cancer lesions, which were strongly positive for immunohistochemical staining. In 69 cases of papillary carcinoma, GPC3 was expressed at an early stage, suggesting that GPC3 expression in thyroid cancer is an early event in developing papillary carcinoma. Further studies are required to determine biological functions and molecular mechanisms underlying the upregulation of GPC3 in thyroid cancer.

Expression of the Glypican-3 Gene in α-fetoprotein-negative Human Hepatocellular Carcinoma

Objective: To investigate the expression of Glypican-3 gene in α-fetoprotein (AFP)-negative hepatocellular carcinoma (HCC) and clarify whether Glypican-3 expression is a useful parameter for the diagnosis of hepatocellular carcinoma (HCC), especially for AFP-negative ones. Methods: Forty-one specimens of AFP-negative hepatocellular carcinoma and para-carcimoma tissue were studied for the expression of Glypican-3 by Northern blot. The expression of Glypican-3 protein was detected immunohistochemically with specific polyclonal antibody. Results: Northern blot analysis indicated that the expression of Glypican-3 mRNA was intensively detected in 30 of 41 AFP-negative HCC (73.17%). In contrast, Glypican- 3 mRNA was only weakly detected in 4 of the surrounding non-tumor liver tissues. There was significant difference in the Glypican-3 mRNA expression in large tumors (>5 cm) (79.31%) and in small tumors (≤5 cm) (41.67%) (P<0.01). Glypican-3 mRNA was more frequently overexpressed in poorly differentiated HCC than in well differentiated ones (76.47% vs. 42.86%, P<0.05). The Glypican-3 expression was not correlated with age, sex, HbsAg seropositivity, fibrocapsule, portal venous embolus and intrahepatic metastasis. The overexpression of Glypican-3 protein in HCC was targeted in tumor cells, not in bile duct cells and other interstitial cells. Conclusion: Glypican-3 was specially overexpressed in AFP-negative HCC, and its expression was closely correlated with the tumor size and tumor grade. Glypican-3 gene may play important roles in hepatocarcinogenesis, and can be used as a new biochemical marker of HCC, especially for AFP-negative HCC.

Usefulness of the Novel Oncofetal Antigen Glypican-3 for Diagnosis of Hepatocellular Carcinoma and Melanoma

Glypican-3 (GPC3) mRNA and protein are expressed in >80% of human hepatocellular carcinomas (HCC) but not in normal tissues except for placenta and fetal liver. The oncofetal antigen GPC3 is a glycosylphosphatidyl inositol-anchored membrane protein and may be secreted. It is a novel tumor marker for human HCC: GPC3 protein was present in sera from 40-50% of HCC patients, but was not detected in sera from patients with liver cirrhosis or chronic hepatitis, or in sera from healthy individuals. alpha-Fetoprotein (AFP) and PIVKA-II (protein induced by vitamin K absence or antagonist-II), are well known major tumor markers for HCC. Generally, AFP shows high positivity for HCC but also high false-positivity in detection assays. Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein (AFP-L3) is a recently described marker of HCC. Detection of AFP-L3 shows a much higher specificity than AFP, but a lower sensitivity. On the other hand, detection of PIVKA-II shows a lower false-positivity, but is not always sensitive enough to detect low levels secreted by small HCCs. There was no correlation between the three tumor markers, AFP, PIVKA-II, and GPC3 in terms of their presence in HCC cells. All three tumor markers showed similar positivity in patients with HCC, detecting 80% of patients with the disease. GPC3 is also a novel tumor marker for the diagnosis of human melanoma, especially in the early stages of the disease. Expression of GPC3 mRNA and protein was evident in tumor cells from >80% of patients with melanoma and melanocytic nevus, which is a common benign lesion. GPC3 protein was detected in sera from 40% (36/91) of melanoma patients, but not in sera from those with large congenital melanocytic nevus, or from healthy donors. Surprisingly, we detected serum GPC3 even in patients with stage 0, in situ melanoma. The positive detection rate of serum GPC3 at stage 0, I, and II (44.4%, 40.0%, 47.6%, respectively) was significantly higher than that of 5-S-cysteinyldopa, a well known tumor marker for melanoma (0.0%, 8.0%, and 10.0%, respectively). Interestingly, GPC3 was highly immunogenic in mice and elicited effective anti-tumor immunity with no evidence of autoimmunity. Thus, GPC3 is useful for diagnosis of HCC and melanoma and may also have a role in immunotherapy or tumor prevention. However, studies in humans are warranted.

Glypican-3 expression is markedly decreased in human gastric cancer but not in esophageal cancer

Background: Deregulation of the expression of glypican-3, a heparan sulfate proteoglycan, has been demonstrated in several human cancers. Methods: In the present study, glypican-3 mRNA expression was analyzed by Northern blotting and in situ hybridization in 20 normal and 41 cancerous esophageal specimens as well as in 15 normal and 32 cancerous gastric tissues. Results: Glypican-3 mRNA was expressed in both normal and esophageal cancer tissues without a significant difference between normal and cancerous tissues, and without a correlation with histological type, tumor stage, tumor grade, or patient survival. Moderate to strong glypican-3 mRNA signals were found in the cytoplasm of squamous epithelial cells of the normal esophagus. In both squamous and adenocarcinomas of the esophagus glypican-3 mRNA signals were also moderately to strongly present in the cytoplasm of the cancer cells. In gastric tissues, glypican-3 mRNA was present in 53% of normal gastric tissue samples, but was below the detection level in all examined gastric cancer samples. Glypican-3 mRNA signals were moderately to strongly present in the cytoplasm of gastric mucosal epithelial cells, but were only very faintly present in some cancer cells. Conclusions: Glypican-3 may be involved in the growth control of normal esophageal and gastric epithelial cells. Furthermore, our results suggest that glypican-3 may play a tumor suppressor role in gastric but not in esophageal cancer.