Association of glypican-3 expression with growth signaling molecules in hepatocellular carcinoma

AIM:To clarify the association of glypican-3(GPC3)expression with Wnt and other growth signaling molecules in hepatocellular carcinoma(HCC). METHODS:Expression of GPC3,Wnt,matrix metalloproteinases(MMPs),sulfatase(SULF)1,SULF2,and other growth signaling molecules was analyzed in HCC cell lines and tissue samples by real-time reverse transcriptionpolymerase chain reaction,immunoblotting,and/or immunostaining.Expression of various genes in GPC3 siRNA-transfected HCC cells was analyzed. RESULTS:GPC3 was overexpressed in most HCCs at mRNA and protein levels and its serum levels weresignificantly higher in patients with HCC than in non- HCC subjects(P<0.05).Altered expressions of various MMPs and growth signaling molecules,some of which were correlated with GPC3 expression,were observed in HCCs.Down-regulation of GPC3 expression by siRNA in GPC3-overexpressing HCC cell lines resulted in a significant decrease in expressions of MMP2,MMP14,fibroblast growth factor receptor 1,insulin-like growth factor 1 receptor.GPC3 expression was significantly correlated with nuclear/cytoplasmic localization ofβ-catenin. CONCLUSION:These results suggest that GPC3,in conjunction with MMPs and growth signaling molecules, might play an important role in the progression of HCC.

Prognostic and clinicopathological significance of glypican-3 overexpression in hepatocellular carcinoma: A meta-analysis

AIM:To investigate the prognostic and clinicopathological significance of glypican-3(GPC3)overexpression in hepatocellular carcinoma(HCC).METHODS:Publications were searched using PubMed,EMBASE,the Cochrane Library and the Chinese Biomedical Literature Database up to March 2013.Inclusion and exclusion criteria were established to screen eligible studies for meta-analysis.The hazard ratios(HRs)of the eligible studies were pooled using RevMan5.2 software to evaluate the impact of GPC3 overexpression on overall survival(OS)and disease-free survival(DFS)in HCC patients.The correlation between GPC3 expression and clinicopathological parameters of HCC was also analyzed.RESULTS:A total of five studies with 493 patients were included in the meta-analysis.The combined HRs indicated that GPC3 overexpression can predict poorOS(n=362 in 3 studies,HR=2.18,95%CI:1.47-3.24,Z=3.86,P=0.0001)and DFS(n=325 in 3 studies,HR=2.05,95%CI:1.43-2.93,Z=3.94,P<0.0001)in HCC patients without heterogeneity.Egger’s and Begg’s tests were applied to detect publication bias,and the results showed that there was no evidence of publication bias detected in the OS studies(the P value for Egger’s test was 0.216)or DFS studies(the P value for Egger’s test was 0.488).The combined odds ratios(ORs)suggested that GPC3 expression tends to be associated with tumor vascular invasion(OR=2.74,95%CI:1.15-6.52,P=0.02),hepatic cirrhosis(OR=2.10,95%CI:1.31-3.36,P=0.002),poor tumor differentiation(OR=0.22,95%CI:0.13-0.40,P<0.00001)and advanced TNM stage(OR=0.31,95%CI:0.18-0.51,P<0.00001).CONCLUSION:From this study,we conclude that GPC3 overexpression tends to be associated with a poor prognosis(poor OS or DFS)in HCC.

Role of Serum Glypican-3 in the Diagnosis of Hepatocellular Carcinoma in the Upper Egypt

Background and Aim: Early diagnosis of hepatocellular carcinoma (HCC) is essential for achieving good prognosis. Glypican 3 (GPC3) has been reported to be raised in HCC in comparison with non-neoplastic lesions. This work aimed to study the role of GPC3 in the early diagnosis of HCC in post-chronic hepatitis C (CHC) cirrhotic patients. Patients and Methods: A comparative study included 60 patients, 40 patients with HCC (HCC group) and 20 patients of CHC without HCC (control group). Diagnosis of HCC was based on abdominal ultrasound and triphasic CT, while biopsy was performed in debating cases. Serum samples for measurement of GPC3 and AFP levels were obtained from all participants. Results: The median levels of both AFP and GPC3 were significantly higher among HCC cases compared to controls. Analysis of the ROC curve showed that both AFP and GPC3 could be used to differentiate HCC cases from controls. AUROCs of GPC3 and AFP were 0.928 and 0.727 respectively, and both were statistically significant with p-values Conclusion: Serum GLP-3 is highly sensitive and specific for detecting HCC, more than AFP for the early detection of HCC, and the combination of both yielded improved sensitivity.

Analysis of in vivo patterns of caspase 3 gene expression in primary hepatocellular carcinoma and its relationship to p21^(WAF1) expression and hepatic apoptosis

AIM To detect the expression of caspase 3gene in primary human hepatocellular carcinoma（HCC）and investigate its relationship to p21WAF1gene expression and HCC apoptosis.METHODS In situ hybridization was employedto determine caspase 3 and p21WAF1expression inHCC.In situ end-labeling was used to detecthepatocytic apoptosis in HCC.RESULTS Twenty-one of 39（53.8%）cases ofHCC were found to express caspase 3transcripts,while 45.2% of HCC failed toexpress caspase 3.Non-cancerous adjacent livertissues showed more positive caspase 3（87.5%,7/8）as compared with HCC（P【0.05）.The expression of caspase 3 is correlated withHCC differentiation,72.2%（13/18）ofmoderately to highly differentiated HCC showedcaspase 3 transcripts positive,while only 38.1%of poorly differentiated HCC harbored caspase 3transcripts（P【0.05）.No relationship wasfound between caspase 3 expression and tumorsize or grade or metastasis,although 52.5%（5/8）of HCC with metastasis were caspase 3positive and a little higher than that with nometastasis（51.6%,P】0.05）.Expression of caspase 3 alone did not affect the apoptosisindex（AI）of HCC.The AI was 7.12%o in caspase3-positive tumors（n=21）,while in caspase 3-negative cases（n=18）6.59%0（P】0.05）.Expression of caspase 3 clearly segregated withp21WAF1positive tumors as compared withp21WAF1-negative cases（16 of 23,69.6% versus5 of 16,31.3%）with statistical significance（P=0.017）.In the cases with positive caspase 3and negative p21WAF1,the Al was found slightlyhigher,but with no statistical significance,thanthat with expression of p21WAF1and caspase 3（7.21‰ vs 6.98‰,P】0.05）.CONCLUSION Loss of caspase 3 expressionmay contribute to HCC carcinogenesis,althoughthe expression of caspase 3 does not correlatewell with cell apoptosis in HCC.p21WAF1may bemerely one of the inhibitors which can reducecaspase 3 mediated cell apoptosis in HCCs.

Low glucose metabolism in hepatocellular carcinoma with GPC3 expression

AIM To investigate the relationship between glucose metabolism and glypican-3(GPc3)expression in hepatocellular carcinoma(Hcc).METHODSImmunohistochemical staining of pathological samples for GPc3 and glucose transporter 1(GLUT1),and whole-body ^(18)F-FDG PET/c T for measuring tumour glucose uptake were performed in 55 newly diagnosed Hcc patients.The maximum standard uptake value(s UVmax)and tumour-to-non-tumourous liver uptake(T/NT)ratio were used to quantify ^(18)F-FDG uptake.In vitro ^(18)F-FDG uptake assay of GPc3-expressing Hep G2 and non-GPc3-expressing RH7777 cel ls was used to examine the effect of GPc3 in cellular glucose metabolism.The relationships between GPc3 expression and ^(18)F-FDG uptake,GLUT1 expression,tumour differentiation,and other clinical indicators were analysed using spearman rank correlation,univariateand multiple logistic regression analyses.RESULTSPositive GPc3 expression was observed in 67.3%of Hcc patients,including 75.0%of those with well or moderately differentiated Hcc and 36.4%of those with poorly differentiated Hcc.There was an inverse relationship between GPc3 expression and s UVmax(Spearman correlation coefficient=-0.281,P=0.038)and a positive relationship between GLUT1 expression and sU Vmax(Spearman correlation coefficient=0.681,P<0.001)in patients with Hcc.Univariate analysis showed that two glucose metabolic parameters(sU Vmax and T/NT ratio),tumour differentiation,lymph node metastasis,and TNM stage were all significantly associated with GPc3 expression(P<0.05),whereas GLUT1 expression,sex,age,tumour size,intrahepatic lesion number,and distant metastasis showed no statistical association(P>0.05).Further multivariate analysis revealed that only the T/N ratio was significantly correlated with GPC3 expression in patients with Hcc(P<0.05).In vitro assay revealed that the uptake of ^(18)F-FDG in GPc3-expressing HepG2 cells was significantly lower than that of non-GPc3-expressing RH7777 cells(t=-20.352,P<0.001).CONCLUSIONThe present study demonstrated that GPc3 expression is inversely associated with glucose metabolism,suggesting that GPc3 may play a role in regulating glucose metabolism in Hcc.

Diagnostic value of glypican-3 in serum and liver for primary hepatocellular carcinoma

AIM:To evaluate the diagnostic value of glypican-3(GPC3) in serum and liver for primary hepatocellular carcinoma(HCC).METHODS:Serum levels of GPC3 and α-fetoprotein(AFP) were measured in 75 patients with primary HCC and 32 patients with liver cirrhosis.Expression of GPC3 and AFP in 58 HCC and 12 cirrhotic specimens was detected with immunohistochemical staining.RESULTS:When the cut-off value of serum GPC3 was set at 300 ng/L,its sensitivity and specificity for HCC were 47.0% and 93.5%,respectively.Among the 14 patients with HCC at stage according to the Barcelona Clinic Liver Cancer staging system,the serum GPC3 level was higher than 300 ng/L in 50%(7/14) patients,the serum AFP level was not ≥ 400 μg/L in any patient.Combined serum AFP and GPC3 significantly increased the sensitivity to the diagnosis of HCC.The GPC3 expression was detected in cytoplasm of HCC cells but not in hepatocytes and bile ducts of benign tumors.Among the 58 HCC patients,the GPC3 was expressed in 100%(28/28) patients with their serum AFP level ≥ 400 μg/L,and in 90%(27/30) patients with their AFP level < 400 μg/L,respectively.The GPC3 was weakly or negatively expressed in all paracarcinomatous and cirrhotic tissue samples.AFP positive HCC cells were only found in 1 out of the 58 HCC patients.CONCLUSION:GPC3 protein is a sensitive and specific serum marker for diagnosis of early HCC.Its expression in liver tissues can be used to discriminate tumor cells from benign hepatic cells.

Glypican-3 is a prognostic factor and an immunotherapeutic target in hepatocellular carcinoma

Glypican-3(GPC3)is a cell surface oncofetal proteoglycan that is anchored by glycosylphosphatidylinositol.Whereas GPC3 is abundant in fetal liver,its expression is hardly detectable in adult liver.Importantly,GPC3is overexpressed in hepatocellular carcinoma(HCC),and several immunohistochemical studies reported that overexpression predicts a poorer prognosis for HCC patients.Therefore,GPC3 would serve as a useful molecular marker for HCC diagnosis and also as a target for therapeutic intervention in HCC.Indeed,some immunotherapy protocols targeting GPC3 are under investigations;those include humanized anti-GPC3cytotoxic antibody,peptide vaccine and immunotoxin therapies.When considering the clinical requirements for GPC3-targeting therapy,companion diagnostics to select the appropriate HCC patients are critical,and both immunohistochemical analysis of tissue sections and measurement of serum GPC3 level have been suggested for this purpose.This review summarizes current knowledge regarding the clinical implication of GPC3detection and targeting in the management of patients with HCC.

Glypican-3 is a biomarker and a therapeutic target of hepatocellular carcinoma

BACKGROUND： The carcinogenesis of hepatocellular car- cinoma （HCC） is a multi-factorial, multi-step and complex process. Early diagnosis and effective treatments are of utmost importance. This review summarized the recent studies of on- cofetal glypican-3 （GPC-3）, a membrane-associated heparan sulfate proteoglycan, in the diagnosis and treatment of HCC. DATA SOURCES： English-language reports published from Iune 2001 to September 2014 were searched from MEDLINE. The key words searched included： GPC-3, biomarker, target and HCC. The sensitivity, specificity, positive and negative predictive values were extracted, and the effect of GPC-3 tar- geted therapy on HCC was also evaluated. RESULTS： GPC-3 plays a crucial role in HCC cell prolifera- tion and metastasis. It mediates oncogenesis involving signal- ing pathways during hepatocyte malignant transformation. GPC-3 expression is increased in atypical hyperplasia and cancerous tissues. GPC-3 levels in HCC patients are related to HBV infection, TNM stage, periportal cancerous embolus, and extrahepatic metastasis. The diagnostic accuracy of the combination of serum GPC-3 and alpha-fetoprotein in HCC is up to 94.3%. Down-regulation of GPC-3 with specific siRNA or anti-GPC-3 antibody alters cell migration, metastasis and invasion behaviors. The nude mice xenograft tumor growth is inhibited by silencing GPC-3 gene transcription.CONCLUSION： Oncofetal GPC-3 is a highly specific biomark- er for the diagnosis of HCC and a promising target molecule for HCC gene therapy.

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

BACKGROUND: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.METHODS: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.RESULTS: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).CONCLUSION:Abnormal oncofetal antigen GPC-3 and GPC-3 mRNA expression in hepatocarcinogenesis may be promising molecular markers for early diagnosis of HCC.

Expression characteristics and diagnostic value of annexin A2 in hepatocellular carcinoma

AIM:To investigate the characteristics and diagnostic value of annexin A2(ANXA2) expression in cancerous tissues and sera of patients with hepatitis B virus(HBV)-related hepatocellular carcinoma(HCC).METHODS:Levels of liver ANXA2 gene transcription or protein expression were analyzed in HCC-,their selfcontrolled precancerous-,and distant cancerous-tissues from 30 HCC.Serum levels of ANXA2 expression in 115 patients with HCC,25 with metastatic liver can-cer,35 with chronic hepatitis,28 with acute hepatitis,38 with cirrhosis,and 30 healthy controls were determined.Clinicopathological characteristics of circulating ANXA2 expression were analyzed,and its diagnostic efficiency and clinical values in HCC were evaluated.RESULTS:ANXA2 expression was localized in both cell membrane and cytoplasm in HCC tissue,mainly in the cytoplasm of matched adjacent cancerous tissue,and there was almost no positive staining in matched distant cancerous tissue.Abnormal expression of liver ANXA2 was present in HCC tissues compared with self-controlled adjacent-and distant-cancerous tissues at protein or mRNA level.Circulating ANXA2 in HCC patients was significantly higher than that of other liver diseases(P < 0.01) except metastatic liver cancer.If the diagnostic cutoff value of ANXA2 level was more than 18 ng/mL,the incidence of serum ANXA2 was 86.96% in the HCC group,80% in the metastatic liver cancer group,31.58% in the liver cirrhosis group,none in the chronic hepatitis or acute hepatitis or normal control group,respectively.Serum ANXA2 expression in HCC patients was correlated with HBV infection(27.38 ± 5.67 ng/mL vs 18.58 ± 7.83 ng/mL,P < 0.01),extrahepatic metastasis(26.11 ± 5.43 ng/mL vs 22.79 ± 5.64 ng/mL,P < 0.01),and portal vein thrombus(26.03 ± 5.99 ng/mL vs 23.06 ± 5.03 ng/mL,P < 0.01),and was significantly higher(P < 0.01) in the moderately-(26.19 ± 5.34 ng/mL) or the poorly-differentiated group(27.05 ± 5.13 ng/mL) than in the well differentiated group(20.43 ± 4.97 ng/mL),and in the tumor node metastasis stages ⅢⅣ(P < 0.01) than in stages ⅠⅡ.ANXA2 was not correlated with patient sex,age,size or-fetoprotein(AFP) level.Area under the receiver operating characteristic curve for the whole range of sensitivities and specificities was 0.796 for ANXA2 and 0.782 for AFP.Combining detection of serum ANXA2 and AFP substantially improved the diagnostic efficiency(96.52%) and the negative predictive value(96.61%) for HCC.CONCLUSION:The characteristics and distributionof ANXA2 expression has good diagnostic potential for HCC diagnosis.

Prognostic value of glypican-3 in patients with HBV-associated hepatocellular carcinoma after liver transplantation

BACKGROUND：Glypican-3（GPC-3）is frequently overexpressed in hepatocellular carcinoma（HCC）.Recent studies have shown that GPC-3 is a highly efficient diagnostic biomarker of HCC and an indicator of poor prognosis in HCC patients who have undergone hepatectomy.However,its prognostic value in patients with HBV-associated HCC after liver transplantation（LT）is not clear.The present study is to evaluate the prognostic value of GPC-3 in patients with HBV-associated HCC after LT.METHODS： A cohort of 104 HCC patients with HBV-associ- ated cirrhosis who had undergone LT at our hospital between 2002 and 2011 were enrolled in this study. Samples of HCC were taken from these patients. GPC-3 protein expression was detected in paraffin-embedded specimens using immunohis- tochemistry. All related clinical data were obtained from the China Liver Transplant Registry. The relationship between GPC-3 expression and clinicopathological parameters was analyzed. Univariate and multivariate Cox-regression analyses were used to identify risk factors for poor prognosis. RESULTS： GPC-3 was expressed in samples from 74 （71.2%） of the 104 patients. GPC-3 was expressed only in HCC cells. Positive staining was correlated with tumor size （P=0.004）, encapsulation （P=0.018）, pathological stage （P--0.027）, portal vein invasion （P=0.043）, tumor differentiation （P=0.002） and the Milan criteria （P=0.016）. The 5-year survival rate and dis- ease-free survival rate of patients with GPC-3-positive were lower than those （38.2% vs 75.4%, P〈0.001; 30.8% vs 69.7%, P=0.001） of patients with GPC-3-negative. Multivariate Coxregression analysis revealed that GPC-3 was an independent risk factor for 5-year survival rate （P=0.031） and disease-free survival rate （P=0.047）, together with tumor differentiation, Milan criteria and pre-operative alpha-fetoprotein.CONCLUSION： GPC-3 is a potential biomarker for poor prognosis after LT in HCC patients with HBV-associated cirrhosis.

Glypican-3-specific cytotoxic T lymphocytes induced by human leucocyte antigen-A*0201-restricted peptide effectively kill hepatocellular carcinoma cells in vitro

Objective: To investigate potential human leucocyte antigen(HLA)-A2-restricted epitope peptides of glypican-3(GPC3) and determine the cytotoxicity of peptidespecific cytotoxic T lymphocytes(CTLs) against hepatocellular carcinoma(HCC) cells.Methods: The potential HLA-A*0201-restricted GPC3 peptides were screened using computer algorithms, T2 cell-binding affinity and stability of peptide/HLA-A*0201 complex assay. The peptide-specific CTLs were generated and their cytotoxicity against GPC3+SMMC 7721 and Hep G2 cells was detected using IFN-g based enzymelinked immunospot and lactate dehydrogenase release assays in vitro.Results: A total of six peptides were identified for bindings to HAL-A2 and the GPC3522–530 and GPC3 229–237 peptides with HLA-A*0201 molecules displayed high binding affinity and stability. The CTLs induced by the GPC3 522–530 or positive control GPC3 144–152 peptide responded to the peptide by producing IFN-g, which were abrogated by treatment with anti-HLA-A2 antibody. The GPC3 522–530-specific CTLs responded to and killed SMMC 7721 and Hep G2 cells, instead of GPC3-silenced SMMC7721 or Hep G2 cells. GPC3 522–530-specific CTLs response to HCC cells was blocked by anti-HLA-A2 antibody.Conclusions: The GPC3 522–530 peptide contains antigen-determinant and its specific CTLs can effectively kill HCC in a HLA-A2-restricted and peptide-dependent manner. Our findings suggest that this peptide may be valuable for development of therapeutic vaccine.

Glypican-3 versus alpha-fetoprotein as a biomarker for hepatocellular carcinoma: a diagnostic meta-analysis

Objective:To assess the diagnostic accuracy of serum GPC3 versus alpha-fetoprotein(AFP)for HCC by using the method of system review.Methods:PubMed and EMBASE were searched from its inception to 20,April 2014 for studies that compared diagnostic accuracy of serum GPC3 with AFP for HCC.Sensitivity,specificity and other measures were pooled using random-effects models.Summary receiver operating characteristic curve analysis was used to summarize the overall test performance.Results:Fourteen studies were included in this meta-analysis.Summary estimates for serum GPC3 and AFP in diagnosing HCC were as follows:sensitivity,69%(95%confidence interval(CI),56-80%)vs.60%(95%CI,50-69%);specificity,91%(95%CI,76-97%)vs.92%(95%CI,84-98%);diagnostic odds ratio(DOR),22(95%CI,6-83)vs.18(95%CI,8-41);and area under sROC,0.85(95%CI,0.81-0.88)vs.0.80(95%CI,0.76-0.83).The pooled sensitivity and specificity for(GPC3+AFP)and AFP were:sensitivity 80%(95%CI,75-85%)vs.64%(95%CI,53-73%)and specificity 86%(95%CI,74-93%)vs.96%(95%CI,86-99%).A significant heterogeneity was found among the ten studies,and meta-regression and subgroup meta-analysis suggested that race and assay type were probably responsible for the heterogeneity.Conclusions:Serum GPC3 may be a promising diagnostic marker of HCC and it was helpful for early detection of primary hepatocellular carcinoma when combined with AFP.More studies for specific race of patients,and using certain methods for detecting GPC3 are required to further confirm the diagnostic value of GPC3 for HCC.

Value of miR-1271 and glypican-3 in evaluating the prognosis of patients with hepatocellular carcinoma after transcatheter arterial chemoembolization

BACKGROUND Hepatocellular carcinoma(HCC)is the third leading cause of cancer death,causing about 750000 deaths worldwide every year.Patients with advanced hepatocellular carcinoma will often only receive transcatheter arterial chemoembolization(TACE).Glypican-3(GPC3)is one of the most promising serum markers for HCC.Abnormal expression of miRNAs may be involved in the occurrence and development of tumor.AIM To explore the value of miR-1271 and GPC3 in evaluating the prognosis of patients with HCC after TACE.METHODS From January 2016 to December 2018,162 patients with advanced HCC who received TACE in our hospital were selected into the cancer group,and 162 patients who underwent physical examination during the same period were selected into the health group.The patients in the HCC group were treated with TACE.The changes of serum GPC3 and circulating miR-1271 in the HCC before and after TACE were analyzed.The expression of serum GPC3 was detected by enzyme-linked immunosorbent assay,and the expression of circulating miR-1271 was detected by real-time quantitative polymerase chain reaction.The methodological results of sensitivity,specificity,and accuracy of miR-1271 and GPC3 alone and joint detection of HCC were also evaluated.RESULTSThe level of serum GPC3 in patients with HCC was significantly higher than that in healthy controls.GPC3 levels were increased in both HCC patients and those treated with TACE compared with healthy controls.After TACE,the level of serum GPC3 was significantly lower than that before treatment(P<0.05),and the level of circulating miR-1271 was significantly higher than that before treatment(P<0.05).There were 112 cases(69.14%)with remission(complete remission+complete remission+stable disease)and 50 cases(30.86%)with relapse disease progression in HCC patients.After TACE,the miR-1271 level in patients with remission and relapse was lower than that in the healthy group,and the GPC3 level was higher than that in the healthy group,the differences were statistically significant(P<0.05).The miR-1271 of relapsed patients was lower than that of remission patients,and the level of GPC3 was higher than that of remission patients,and the difference was statistically significant(P<0.05).The sensitivity of combined detection of miR-1271 and GPC3 was significantly higher than that of single detection,and the difference was statistically significant(P<0.05);while the specificity of the two combined detections was lower than that of the single detection;and the accuracy was slightly higher than that of single detection,but the difference was not statistically significant.CONCLUSION The level of miR-1271 in patients with HCC was significantly increased and the level of GPC3 was decreased after TACE.Monitoring the levels of serum GPC3 and circulating miR-1271 has important clinical reference value for evaluating the prognosis of patients with HCC.The levels of serum GPC3 and circulating miR-1271 may help to determine tumor recurrence,evaluate survival status,and guide the next step of treatment.

New Immunological Approaches in Hepatocellular Carcinoma: Glypican-3 (GPC-3) Opportunities and Challenges

Immunotherapy is one of the strategies to boost natural defenses to fight cancer. Immuno-oncology is an artificial stimulation of the human immune system to recognize and kill selectively neoplastic cells at different stage of transformation. Cancer cells have tumor antigens and the antibody of the immune system, binding them, can detect molecules on their extracellular side of cell membrane. Among these proteins, it is rising in interest and used for early detection of hepatocellular carcinoma (HCC) Glypican-3 (GPC-3) protein. It is a heparan sulfate proteoglycan (HSPG), anchored to the cell membrane of transformed hepatocytes. We investigated its function as key regulator of hepatocytes neoplastic transformation. Noteworthy, GPC-3 protein has been implicated in different pathways from cell growth to cell motility and migration. More recently, GPC-3 has been evaluated as a useful marker for HCC due to its increased expression in the liver during tumorigenesis and its absence in normal liver. Immunotherapy that targets GPC-3 domains and its connected proteins are currently under investigation. These new biomarkers may hold potential for the detection and treatment of HCC and other diseases in which GPC-3 may be overexpressed and/or play a crucial role. This review will summarize the current knowledge regarding the active immunotherapy developed to treat HCC and it will evaluate aspects of GPC-3 (structure and biology) as advantages and potential pitfalls for considering it as a valuable immunotherapeutic target. We also elaborated the current literature with the aim to better understand its biological interactions at a molecular and cellular level to identify alternative or combined targets, due to the existing gap in the literature surrounding GPC-3. The role GPC-3 plays in the hepatocellular carcinoma phenotype can be targeted for a novel immunotherapy strategy that can specify cell-mediated destruction of neoplastic cell that spares normal liver tissue, and it can be exploited as a new serum marker to trend for diagnosis and disease progression measurements. We believe further investigation of its functions and structure, including alternative cellular localizations, is necessary to evaluate GPC-3 as valuable target to cure this cancer.

Tissue inhibitor of metalloproteinase-3 expression affects clinicopathological features and prognosis of aflatoxin B1-related hepatocellular carcinoma

BACKGROUND The dysregulation of tissue inhibitor of metalloproteinase-3(TIMP3)was positively correlated with the progression of hepatocellular carcinoma(HCC).However,it is not clear whether TIMP3 expression is associated with the clinico-pathological features and prognosis of aflatoxin B1(AFB1)-related HCC(AHCC).A retrospective study,including 182 patients with AHCC,was conducted to explore the link between TIMP3 expression in cancerous tissues and the clinico-pathological characteristics and prognosis of AHCC.TIMP3 expression was detected by immunohistochemistry and its effects on the clinicopathological features and prognosis of AHCC were evaluated by Kaplan-Meier survival analysis and Cox regression survival analysis.Odds ratio,hazard ratio(HR),median overall survival time(MST),median tumor recurrence-free survival time(MRT),and corresponding 95%confidential interval(CI)was calculated to RESULTS Kaplan-Meier survival analysis showed that compared with high TIMP3 expression,low TIMP3 expression in tumor tissues significantly decreased the MST(36.00 mo vs 18.00 mo)and MRT(32.00 mo vs 16 mo)of patients with AHCC.Multivariate Cox regression survival analysis further proved that decreased expression of TIMP3 increased the risk of death(HR=2.85,95%CI:2.04-4.00)and tumor recurrence(HR=2.26,95%CI:1.57-3.26).Furthermore,decreased expression of TIMP3 protein in tissues with AHCC was significantly correlated with tumor clinicopatho-logical features,such as tumor size,tumor grade and stage,tumor microvessel density,and tumor blood invasion.Additionally,TIMP3 protein expression was also negatively associated with amount of AFB1-DNA adducts in tumor tissues.CONCLUSION These findings indicate that the dysregulation of TIMP3 expression is related to AHCC biological behaviors and affects tumor outcome,suggesting that TIMP3 may act as a prognostic biomarker for AHCC.

Values of circulating GPC-3 mRNA and alpha-fetoprotein in detecting patients with hepatocellular carcinoma

BACKGROUND: The prognosis of hepatocellular carcinoma (HCC) is poor and its early diagnosis is of the utmost importance. This study aimed to investigate the values of glypican-3 (GPC-3) expression in the liver and sera and its gene transcription for diagnosis and monitoring of metastasis of HCC. METHODS: Liver GPC-3 was analyzed in HCC tissues from 36 patients by immunohistochemistry and Western blotting. GPC-3 mRNA from circulating peripheral blood mononuclear cells from 123 HCC patients or 246 patients with other diseases or 36 HCC tissues was amplified by RT-PCR, quantitative realtime PCR, and confirmed by DNA sequencing. Circulating GPC-3 level was detected by ELISA. RESULTS: The increasing expression of GPC-3 was observed from non-cancerous to cancerous tissues, with brown granule-like staining localized in tumor parts of atypical hyperplasia and HCC formation. The positive rate of GPC-3 was 80.6% in HCC, 41.7% in their paracancerous tissues, and none in distal cancerous tissues (P【0.001), with no significant difference in differentiation grade and tumor number except for size (Z=2.941, P=0.003). Serum GPC-3 was detected only in HCC (52.8%) and significant difference was found between GPC-3 and tumor size (χ2 =6.318, P=0.012) or HBV infection (χ2 =23.362, P【0.001). Circulating GPC-3 mRNA was detected in 70.7% of HCC tissues, with relation to TNM stage, periportal cancerous embolus, and extra-hepatic metastasis (P【0.001). The combination ofcirculating GPC-3, GPC-3 mRNA and alpha-fetoprotein is of complementary value for HCC diagnosis (94.3%). CONCLUSION: Both GPC-3 overexpression and GPC-3 mRNA abnormality could be used as markers for the diagnosis of HCC and monitoring its metastasis.

血清学标志物甲胎蛋白、PIVKA-Ⅱ和磷脂酰肌醇蛋白聚糖3联合诊断肝癌的meta分析

目的:探讨血清生物标志物甲胎蛋白(AFP)、维生素K缺失或拮抗剂Ⅱ诱导的蛋白质(PIVKA-Ⅱ)和磷脂酰肌醇蛋白聚糖3(GPC-3)单独或联合用于肝细胞癌(以下简称肝癌)诊断的价值。方法:检索PubMed、Web of Science、Embase三个数据库,收集2002年以来发表的AFP、PIVKA-Ⅱ和GPC-3单独或联合用于诊断肝癌的文献。根据纳入和排除标准筛选文献并提取相关数据。利用诊断准确性研究的质量评价(QUADAS)检查表对纳入的文献进行质量评价,并采用Meta DiSc软件、Review Manager 5.4软件和Stata 15.1软件对AFP、PIVKA-Ⅱ和GPC-3单用和联合使用诊断肝癌的受试者工作特征曲线下面积(AUC)、敏感度、特异度等指标进行数据分析。结果:共纳入32篇文献。Meta分析结果显示,单个标志物用于诊断肝癌时,PIVKA-Ⅱ的AUC值最高,为0.88(95%CI:0.85~0.91),其次是GPC-3和AFP;多个标志物联合用于诊断肝癌的AUC均高于单个标志物,其中PIVKA-Ⅱ联合GPC-3诊断的AUC值最高,为0.90(95%CI:0.87~0.92)。单个标志物用于诊断肝癌时,PIVKA-Ⅱ和GPC-3的敏感度相对较高(分别为0.75和0.76),但GPC-3的特异度不如PIVKA-Ⅱ和AFP(AFP、PIVKA-Ⅱ和GPC-3分别为0.87、0.88和0.81);多个标志物联合用于诊断肝癌的敏感度较单个标志物诊断时有所提高,但特异度无明显提高。单个标志物用于诊断肝癌时,PIVKA-Ⅱ的诊断比值比(DOR)最高,为22(95%CI:13~36),其次是GPC-3和AFP;两个标志物联合用于诊断肝癌的DOR均高于单个标志物,其中AFP联合GPC-3诊断的DOR最高,为25(95%CI:9~67);三个标志物联合用于诊断肝癌时的DOR明显降低,为10(95%CI:7~45)。结论:单个标志物用于肝癌诊断时,PIVKA-Ⅱ的诊断价值更高。两种标志物联合能显著提高肝癌诊断的敏感度,三种标志物联合未能进一步提高诊断价值。结合临床实际,推荐AFP联合PIVKA-Ⅱ用于肝癌的诊断。

Clinical significance of serum sB7-H3 and IL-1 in patients with hepatitis B related hepatocellular carcinoma

Objective: To explore the clinical significance of serum sB7-H3 and IL-1 in patients with hepatitis B associated hepatocellular carcinoma. Methods: 122 cases of patients with hepatitis B related liver diseases admitted to our hospital from January 2015 to June 2018 were selected, including 47 cases of patients with hepatitis B related hepatocellular carcinoma, 41 cases of patients with hepatitis B related cirrhosis and 34 cases of patients with hepatitis B virus, and 45 cases of healthy persons in the same period as normal control group. The serum sB7-H3, IL-1α, IL-1β levels were compared among the four groups. The relationship between serum sB7-H3, IL-1α, IL-1β and clinicopathological characteristics was analyzed, and the correlation of sB7-H3, IL-1α and IL-1β was analyzed by Pearson method. The efficacy of serum sB7-H3, IL-1α, IL-1β in early diagnosis and prognostic evaluation for hepatitis B associated hepatocellular carcinoma were analyzed by ROC and Logistic regression analysis. Results: The serum sB7-H3, IL-1α, IL-1β levels in the four groups were in order from high to low: hepatitis B related hepatocellular carcinoma>hepatitis B related cirrhosis>hepatitis B virus>normal control group, all above had statistical difference (P<0.05). The high levels of serum sB7-H3, IL-1α, IL-1β were significantly correlated with TNM stage, alpha fetoprotein level and lymph node metastasis (P<0.05). Spearman correlation analysis results showed that the serum level of sB7-H3 was positively correlated with IL-1α and IL-1β(r=0.837, 0.756;P<0.05), the serum level of IL-1α was positively correlated with IL-1β(r=0.734, P<0.05). The ROC curve and Logistic regression analysis showed that the AUC of sB7-H3, IL-1α, IL-1β and combined detection for the diagnosis of hepatitis B associated hepatocellular carcinoma was 0.893, 0.887, 0.881, 0.961 (P<0.05), respectively;the AUC of sB7-H3, IL-1α, IL-1β and combined detection for the prognostic evaluation for hepatitis B associated hepatocellular carcinoma was 0.843, 0.837, 0.834, 0.917, respectively. Conclusion:Serum sB7-H3, IL-1α, IL-1β levels could all be used for the early diagnosis and prognostic evaluation of hepatitis B associated hepatocellular carcinoma, and the combined detection is more effective, which has important clinical significance.

Glypican-3蛋白在肝细胞癌患者血清和组织中的表达及意义

目的:探讨肝细胞癌(HCC)患者血清和组织中Glypican-3(GPC3)蛋白的表达及临床意义.方法:收集HCC患者27例和肝良性病变患者28例的术前1d空腹血清及组织标本,采用Western blot检测血清中GPC3蛋白,同时采用免疫组织化学SP法检测组织标本中GPC3的表达情况.结果:27例HCC患者HCC组织、癌旁组织和远癌肝组织中GPC3蛋白阳性的表达率分别为81.5%,0%和0%(HC:23.4689,P<0.001),术前血清中GPC3蛋白阳性率分别为55.6%;28例肝良性病变患者组织和血清中阳性率均为0%;GPC3蛋白对HCC诊断的敏感性为55.6%,特异性为100%,误诊率为0%.HCC患者血清中GPC3蛋白表达与其瘤体大小和病理分级之间差异有显著性(P<0.05),而与患者年龄、性别、HBsAg及AFP值之间差异无显著性(P>0.05).结论:GPC3蛋白在HCC患者血清和组织中有较高的表达,对诊断HCC有较高的敏感性和特异性,可作为HCC早期诊断的标志物.