Preoperative prediction of glypican-3 positive expression in solitary hepatocellular carcinoma on gadoxetate-disodium enhanced magnetic resonance imaging.

Purpose: As a coreceptor in Wnt and HGF signaling, glypican-3 (GPC-3) promotes the progression of tumor and is associated with a poor prognosis in hepatocellular carcinoma (HCC). GPC-3 has evolved as a target molecule in various immunotherapies, including chimeric antigen receptor T cell. However, its evaluation still relies on invasive histopathologic examination. Therefore, we aimed to develop an easy-to-use and noninvasive risk score integrating preoperative gadoxetic acid-enhanced magnetic resonance imaging (EOB-MRI) and clinical indicators to predict positive GPC-3 expression in HCC. Methods and materials: Consecutive patients with surgically-confirmed solitary HCC who underwent preoperative EOB-MRI between January 2016 and November 2021 were retrospectively included. EOB-MRI features were independently evaluated by two masked abdominal radiologists and the expression of GPC-3 was determined by two liver pathologists. On the training dataset, a predictive scoring system for GPC-3 was developed against pathology via logistical regression analysis. Model performances were characterized by computing areas under the receiver operating characteristic curve (AUCs). Results: A total of 278 patients (training set, n=156; internal validation set, n=39; external validation set, n=83) with solitary HCC (208 [75%] with positive GPC-3 expression) were included. Serum alpha-fetoprotein >10 ng/ml (AFP, odds ratio [OR]=2.3, four points) and five EOB-MR imaging features, including tumor size >3.0cm (OR=0.5, -3 points), nonperipheral "washout" (OR=3.0, five points), infiltrative appearance (OR=9.3, 10 points), marked diffusion restriction (OR=3.3, five points), and iron sparing in solid mass (OR=0.2, -7 points) were significantly associated with positive GPC-3 expression. The optimal threshold of scoring system for predicting GPC-3 positive expression was 5.5 points, with AUC 0.726 and 0.681 on the internal and external validation sets, respectively. Conclusion: Based on serum AFP and five EOB-MRI features, we developed an easy-to-use and noninvasive risk score which could accurately predict positive GPC-3 HCC, which may help identify potential responders for GPC-3-targeted immunotherapy.

Neuronal Glypican4 promotes mossy fiber sprouting through the mTOR pathway after pilocarpine-induced status epilepticus in mice.

In temporal lobe epilepsy (TLE), abnormal axon guidance and synapse formation lead to sprouting of mossy fibers in the hippocampus, which is one of the most consistent pathological findings in patients and animal models with TLE. Glypican 4 (Gpc4) belongs to the heparan sulfate proteoglycan family, which play an important role in axon guidance and excitatory synapse formation. However, the role of Gpc4 in the development of mossy fibers sprouting (MFS) and its underlying mechanism remain unknown. Using a pilocarpine-induced mice model of epilepsy, we showed that Gpc4 expression was significantly increased in the stratum granulosum of the dentate gyrus at 1 week after status epilepticus (SE). Using Gpc4 overexpression or Gpc4 shRNA lentivirus to regulate the Gpc4 level in the dentate gyrus, increased or decreased levels of netrin-1, SynI, PSD-95, and Timm score were observed in the dentate gyrus, indicating a crucial role of Gpc4 in modulating the development of functional MFS. The observed effects of Gpc4 on MFS were significantly antagonized when mice were treated with L-leucine or rapamycin, an agonist or antagonist of the mammalian target of rapamycin (mTOR) signal, respectively, demonstrating that mTOR pathway is an essential requirement for Gpc4-regulated MFS. Additionally, the attenuated spontaneous recurrent seizures (SRSs) were observed during chronic stage of the disease by suppressing the Gpc4 expression after SE. Altogether, our findings demonstrate a novel control of neuronal Gpc4 on the development of MFS through the mTOR pathway after pilocarpine-induced SE. Our results also strongly suggest that Gpc4 may serve as a promising target for antiepileptic studies.

Does Loss of Immunohistochemical Expression of Glypican 3 in Oral Squamous Cell Carcinoma Play a Role in the Wnt/ß-catenin Signaling Pathway?

Glypican 3 (GPC3) is a cell membrane protein and plays a dual role, as a tumor suppressor and oncogene, depending on its structure. It is known to regulate the Wnt/ß-catenin signaling pathway and affect cell growth and proliferation. ß-catenin plays a major oncogenic role in progression of oral squamous cell carcinoma (OSCC); thus, this study aimed to explore the relationship between ß-catenin and GPC3 in OSCC. Immunoexpression of GPC3 and ß-catenin was evaluated semiquantitatively in tumor tissue (n=80) and normal oral mucosa tissue (n=20). For GPC3, the percentage of stained cells and the staining intensity were assessed. For ß-catenin, the percentage of stained cells, localization, and intensity of staining were assessed at the tumor-invasive front. The Pearson correlation was used to determine the correlation between the GPC3 and ß-catenin immunoreactivity. Significantly decreased expression of GPC3 (P=0.008) and a highly significant difference in the case of localization of ß-catenin (P=0.0001) were observed in OSCC when compared with normal oral mucosa. Cytoplasmic expression with a shift of ß-catenin expression to the nucleus was seen in OSCC in comparison with primarily membranous and membranous and cytoplasmic staining in normal mucosa. A significant difference was observed with respect to localization of stain, with ß-catenin staining moving to the nuclear compartment with an increase in the tumor grade (P=0.011). No correlation was observed between ß-catenin and GPC3 expression in OSCC cases. It is concluded that loss of expression of GPC3 in OSCC compared with normal oral mucosa indicates that it plays the role of a tumor suppressor gene in OSCC and its expression is therefore silenced in OSCC.

Glypican-1, -3, -5 (GPC1, GPC3, GPC5) and Hedgehog Pathway Expression in Oral Squamous Cell Carcinoma.

Proteoglycans are involved in tumor development and may regulate the Hedgehog (HH) pathway. This study aimed to investigate the gene and protein expression of glypican-1 (GPC1), -3 (GPC3), and -5 (GPC5) in oral squamous cell carcinoma (OSCC) and tumor-free lateral margins (TM) and their association with the HH pathway. Quantitative PCR was performed for GPC1, GPC3, GPC5, SHH, PTCH1, SMO, and GLI1 genes in samples of OSCC (n=31), TM (n=12), and non-neoplastic oral mucosa (NNM) of healthy patients (n=6), alongside an immunohistochemical evaluation of GPC1, GPC3, and GPC5 proteins and HH proteins SHH and glioma-associated oncogene homolog 1 (GLI1). Double staining for GPC3/SHH, GPC5/SHH, GPC3/tubulin [ac Lys40], GPC5/Tubulin [ac Lys40], and GPC5/GLI1 was also performed. Overexpression of GPC1 and GPC5 in tumor samples and underexpressed levels of GPC3 gene transcripts were observed when compared with TM (standard sample). HH pathway mRNA aberrant expression in OSCC samples and a negative correlation between GPC1 and GPC5 at transcription levels were detected. GPC1 staining was rare in OSCC, but positive cells were found in NNM and TM. Otherwise positive immunostaining for GPC3 and GPC5 was observed in OSCCs, but not in NNM and TM. Blood vessels adjacent to tumor islands were positive for GPC1 and GPC5. Co-localization of GPC3-positive and GPC5-positive cells with SHH and Tubulin [ac Lys40] proteins was noted, as well as of GPC5 and GLI1. The absence of the GPC1 protein in neoplastic cells, underexpression of the GPC3 gene, and co-localization of GPCs and HH proteins may indicate the maintenance of aberrant HH pathway activation in OSCC.

Clinical Impact of Circulated miR-1291 in Plasma of Patients with Liver Cirrhosis (LC) and Hepatocellular Carcinoma (HCC): Implication on Glypican-3 Expression.

PURPOSE: Liver cirrhosis (LC) is considered to be the end stage of chronic hepatopathies which may lead to hepatocellular carcinoma (HCC). Glypican-3 is one of the most promising serum markers for HCC. Abnormal expression of miRNAs may participate in cancer development and progression. In this study, we aimed to evaluate the relation between the expression of miR-1291 and GPC3 production as a non-invasive tool to differentiate patients with LC and HCC. METHODS: HCV patients (100) were divided into two groups; HCC (I) and LC (II). Fifty hepatitis-free subjects served as the control group (III). Expression of serum GPC3 was performed by enzyme-linked immunosorbent assay, and expression of circulating miR-1291 was performed by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Serum levels of GPC3 were significantly elevated in patients with HCC compared with the LC group. Both groups have increased GPC3 levels in relation to healthy controls. Serum GPC3 levels with a cutoff value of 619.5 pg/ml had a 50% sensitivity and 89.3% specificity while alpha-fetoprotein (AFP) with a cutoff value of 8.5 ng/ml had a higher sensitivity (87.5%) and specificity (100%) in the detection of HCC. The primary use of both markers improved the specificity to 100%. miR-1291 was significantly upregulated in HCC and LC patients compared with control subjects. CONCLUSIONS: Our findings might indicate that miR-1291 exert oncogenic effects in hepatic carcinogenesis through positive regulation of GPC3 expression. We propose that GPC3 overexpression and its associated oncogenic effects are linked to the upregulation of miR-1291 in HCV patients.

Glypican 6 is a putative biomarker for metastatic progression of cutaneous melanoma.

Due to the poor prognosis of advanced metastatic melanoma, it is crucial to find early biomarkers that help identify which melanomas will metastasize. By comparing the gene expression data from primary and cutaneous melanoma samples from The Cancer Genome Atlas (TCGA), we identified GPC6 among a set of genes whose expression levels can distinguish between primary melanoma and regional cutaneous/subcutaneous metastases. Glypicans are thought to play a role in tumor growth by regulating the signaling pathways of Wnt, Hedgehogs, fibroblast growth factors (FGFs), and bone morphogenetic proteins (BMPs). We showed that GPC6 expression was up-regulated in a melanoma cell line compared to normal melanocytes and in metastatic melanoma compared to primary melanoma. Furthermore, GPC6 expression was positively correlated with genes largely involved in cell adhesion and migration in both melanoma samples and in RNA-seq samples from other TCGA tumors. Our results suggest that GPC6 may play a role in tumor metastatic progression. In TCGA melanoma samples, we also showed that GPC6 expression was negatively correlated with miR-509-3p, which has previously been shown to function as a tumor suppressor in various cancer cell lines. We overexpressed miR-509-3p in A375 melanoma cells and showed that GPC6 expression was significantly suppressed. This result suggested that GPC6 was a putative target of miR-509-3p in melanoma. Together, our findings identified GPC6 as an early biomarker for melanoma metastatic progression, one that can be regulated by miR-509-3p.

Expression of RNA-binding protein LIN28 in classic gastric hepatoid carcinomas, gastric fetal type gastrointestinal adenocarcinomas, and hepatocellular carcinomas: An immunohistochemical study with comparison to SALL4, alpha-fetoprotein, glypican-3, and Hep Par1.

INTRODUCTION: Gastric hepatoid carcinomas (GHCs) include type I (classic) and type II (fetal type gastrointestinal adenocarcinoma). The classic type shows overlapping morphologic features with those of hepatocellular carcinoma (HCC). The aim of this study is to investigate expression of LIN28 in GHCs and explore its utility to distinguish classic GHC from HCC. METHODS: We investigated immunohistochemical expression of LIN28 in 93 primary GHCs (47 type I, 46 type II) and 60 HCCs with comparison to SALL4, AFP, glypican-3, Hep Par1, p-CEA and CK7. We also stained LIN28 and SALL4 in 52 conventional gastric adenocarcinomas to assess their specificity in gastric carcinomas. RESULTS: Classic GHCs and fetal type gastrointestinal adenocarcinomas showed positive LIN28 in 21/47 (45%) and 10/46 (22%), SALL4 in 41/47 (87%) and 36/46 (78%), AFP in 30/46 (65%) and 33/46 (72%), glypican-3 in 31/41 (76%) and 24/38 (63%), Hep Par1 in 27/41 (66%) and 28/37 (76%), and CK7 in 15/40 (38%) and 25/38 (66%), respectively. p-CEA staining was seen in 19/44 (43%) classic GHCs. Among HCCs, LIN28, SALL4, AFP, glypican-3, Hep Par1, p-CEA and CK7 was seen in 1/60 (2%), 0/60 (0%), 6/30 (20%), 23/30 (77%), 29/30 (97%), 28/30 (93%) and 21/30 (70%) cases, respectively. LIN28 and SALL4 staining was seen in 2/52 (4%) and 14/52 (27%) gastric conventional adenocarcinomas, respectively. The sensitivity and specificity of distinguishing classic GHCs from HCCs was 45% and 98% for LIN28, 87% and 100% for SALL4, 65% and 80% for AFP, 76% and 30% for glypican-3, 66% and 3% for Hep Par1, 43% and 7% for p-CEA, and 38% and 30% for CK7, respectively. Combining LIN28 and SALL4 increased the sensitivity to 96% with 98% specificity to distinguish classic GHCs from HCCs. CONCLUSIONS: LIN28 is a very specific marker (98% specificity) for distinguishing classic GHCs from HCCs though it is not as sensitive as SALL4. AFP, glypican-3, Hep Par1 and p-CEA are not useful in distinguishing classic GHCs from HCCs. Combining LIN28 and SALL4 increased the sensitivity to distinguish classic PHCs from HCCs.

Glypican-1 immunohistochemistry is a novel marker to differentiate epithelioid mesothelioma from lung adenocarcinoma.

Histological morphology alone is not sufficient for the pathological diagnosis of malignant mesothelioma. Positive and negative immunohistochemical markers are necessary to differentiate it from lung adenocarcinoma. As calretinin and D2-40, the recognized positive markers of mesothelioma, are expressed in lung adenocarcinoma to some extent, novel markers with high specificity are desirable. In this study, we investigated the applicability of glypican-1 immunohistochemistry to differentiate epithelioid mesothelioma from lung adenocarcinoma. We investigated 82 cases of epithelioid mesothelioma and 97 cases of lung adenocarcinoma for glypican-1 expression by immunohistochemistry using a commercially available antibody. All 82 cases of epithelioid mesothelioma showed glypican-1 expression, most with diffuse and strong reactivity. In contrast, only three cases of lung adenocarcinoma showed focal glypican-1 expression. Glypican-1 expression showed 100 sensitivity, 97% specificity, and a 98% accuracy rate to differentiate epithelioid mesothelioma from lung adenocarcinoma. The sensitivity of glypican -1 immunohistochemistry is as high as that of calretinin and D2-40, and its specificity is far better than that of calretinin and D2-40. Therefore, we recommend including glypican -1 immunohistochemistry as a positive marker of epithelioid mesothelioma.

Correlation between HBV protein preS2 and tumor markers of hepatocellular carcinoma.

BACKGROUND: Alpha-fetoprotein (AFP) and Glypican 3 (GPC3) are both oncogenes and reactivated in hepatocellular carcinoma (HCC). PreS2 has been proved to be an important transactivator in HCC. In this study, we aim to provide evidence that HBV protein preS2 is responsible for AFP and GPC3's reactivation in HCC. METHODS: Totally Sixty-three cases of HCC, aged 34-79, who were surgically treated and pathologically confirmed were enrolled. The levels of AFP in peripheral serum were detected with electrochemical luminescence method before surgery. Levels of GPC3 in HCC samples were evaluated by immunohistochemistry. Luciferase reporter assays were used to measure the effect of preS2 on AFP and GPC3 promoters. RESULTS: AFP level and GPC3 but not albumin were significantly higher in preS2-positive HCC samples than preS2-negative HCC samples. And the preS2 protein expression was positively related with serum AFP level and GPC3 expression. Furtherly, dual luciferase assay showed that preS2 activated AFP and GPC3 promoter activity. CONCLUSION: The expression of preS2 protein relates closely to HCC markers AFP and GPC3.

Gallic acid against hepatocellular carcinoma: An integrated scheme of the potential mechanisms of action from in vivo study.

The global burden of hepatocellular carcinoma is increasing; actually, it is estimated as 750,000 new cases annually. This study was initiated to emphasize the possibility that gallic acid could alleviate hepatocarcinogenesis in vivo. In this study, 40 rats were enrolled and distributed as follows; group 1 was set as negative control, while all of groups 2, 3, and 4 were orally received N-nitrosodiethylamine for hepatocellular carcinoma induction. Group 2 was left untreated, whereas groups 3 and 4 were orally treated with gallic acid and doxorubicin, respectively. The current data indicated that gallic acid administration in hepatocellular carcinoma bearing rats yielded significant decline in serum levels of alpha-fetoprotein, glypican-3, and signal transducer and activator of transcription 3 along with significant enhancement in serum suppressors of cytokine signaling 3 level. Also, gallic acid-treated group displayed significant downregulation in the gene expression levels of hepatic gamma glutamyl transferase and heat shock protein gp96. Intriguingly, treatment with gallic acid remarkably ameliorated the destabilization of liver tissue architecture caused by N-nitrosodiethylamine intoxication as evidenced by histopathological investigation. In conclusion, this study demonstrates that the hepatocarcinogenic effect of N-nitrosodiethylamine can be abrogated by gallic acid supplementation owing to its affinity to regulate signal transducer and activator of transcription 3 signaling pathway through its outstanding bioactivities including antioxidant, anti-inflammatory, apoptotic, and antitumor effects.

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

PURPOSE: To correlate expression of Glypican-3 in Wilms tumor with histopathology, stage, and outcome. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Glypican-3 is useful but not superior to Hep Par 1 in differentiating hepatocellular carcinoma from other liver tumours.

To assess the diagnostic utility of glypican-3 (GPC-3) in comparison to Hep Par 1 in the diagnosis of liver tumours, a cross-sectional study involving 66 resected liver tumours were tested for the protein expression of these markers by immunohistochemistry using monoclonal antibodies. Of the 66 cases, 26 (39.4%) were hepatocellular carcinoma (HCC), 4 (6.1%) were intrahepatic cholangiocarcinoma and 36 (54.5%) were metastatic tumours. Hep Par 1 and GPC-3 expressions in HCC were 24/26 (92.3%) and 19/26 (73.1%) respectively. In contrast, of non-HCC cases, only 2/40 cases (5.0%) expressed Hep Par 1, including a metastatic colorectal adenocarcinoma and a metastatic gastric adenocarcinoma. GPC-3 was expressed in 3/40 cases (7.5%), i.e. a metastatic adenocarcinoma of unknown origin, a metastatic gastric adenocarcinoma and an intrahepatic cholangiocarcinoma. The sensitivity and specificity for Hep Par 1 were 92.3% and 95% respectively while that of GPC-3 was 73.1% and 92.5% respectively. GPC-3 is a useful marker in the diagnosis of HCC. However it is not superior to Hep Par 1 in its sensitivity and specificity. We recommend that it is utilized together with Hep Par 1 as a panel in the diagnosis of HCC.

Glypican-3 and KRT19 are markers associating with metastasis and poor prognosis of pancreatic ductal adenocarcinoma.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor with metastasis in most patients at diagnosis. The molecular mechanisms associated with its high malignancy have not been fully elucidated. This study investigated the clinicopathological significances of GPC3 and KRT19 expression in PDAC. METHODS: GPC3, KRT19, and CA19-9 protein expression were measured by immunohistochemistry. RESULTS: GPC3 and KRT19 protein levels were overexpressed in PDAC tumors compared to normal pancreatic tissues, benign pancreatic tissues, and peritumoral tissues (P< 0.01). The percentage of positive GPC3 and KRT19 expression were significantly higher in PDAC patients with larger tumor size, poorly differentiated tumor, lymph node metastasis, invasion, and TNM stage III/IV disease than in patients with small tumor size, well-differentiated tumor, no lymph node metastasis and invasion, as well as TNM stage I/II stage disease (P< 0.05 or P< 0.01). Benign pancreatic lesions with positive GPC3 and KRT19 protein expression exhibited dysplasia or intraepithelial neoplasia. Kaplan-Meier survival analysis showed that PDAC patients with positive GPC3 and KRT19 expression survived significantly shorter than patients with negative GPC3 and KRT19 expression (P < 0.05 or P< 0.001). Cox multivariate analysis revealed that positive GPC3 and KRT19 expression were independent poor prognosis factors in PDAC patients. CONCLUSIONS: GPC3 and KRT19 overexpression are associated with carcinogenesis, progression, and poor prognosis in patients with PDAC and a valuable biomarker for diagnosis of PDAC.

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

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

Gene expression analysis for evaluation of potential biomarkers in hepatocellular carcinoma.

BACKGROUND: The poor prognosis of hepatocellular carcinoma (HCC) and the lack of specific screening markers underline the need for new biomarkers for human hepatocarcinogenesis. MATERIALS AND METHODS: We investigated 10 postulated biomarkers for HCC (AFP, GPC3, OPN, IGF1, HGF, SPINK1, KPNA, FUCA1, CgA, HSP90) with microarray gene expression analysis and real-time polymerase chain reaction (RT-PCR) in HCC tissues of different etiologies. RESULTS: Four candidate genes (FUCA1, HGF, IGF1, CgA) showed low median fold changes (fc) of expression compared to corresponding non-malignant liver tissues (fc range=0.2-0.8; maximum 15% of samples). The classic biomarker, alpha-fetoprotein (AFP), was significantly over-expressed (fc=2.4) in 30% of tumors. High tumor AFP expression was associated with significantly elevated serum AFP concentrations (>90% of cases). Five genes (OPN, SPINK1, GPC3, HSP90, KNPA2) showed significantly higher expression than AFP in 64% to 82% of samples (median fc range=2.9-8.3). RT-PCR analyses gave similar results. CONCLUSION: Unlike previous studies, our results did not confirm FUCA1, HGF, IGF1 or CgA as potential markers for HCC. In contrast, OPN, SPINK1, GPC3 and KNPA2 were significantly over-expressed in HCC tissues. These genes may be useful in developing future biomarkers and therapeutic strategies for HCC.

Tumor-promoting/progressing role of additional chromosome instability in hepatic carcinogenesis in Sgo1 (Shugoshin 1) haploinsufficient mice.

A major etiological risk factor for hepatocellular carcinoma (HCC) is infection by Hepatitis viruses, especially hepatitis B virus and hepatitis C virus. Hepatitis B virus and hepatitis C virus do not cause aggressive activation of an oncogenic pathway, but they transactivate a broad array of genes, cause chronic inflammation, and, through interference with mitotic processes, lead to mitotic error-induced chromosome instability (ME-CIN). However, how ME-CIN is involved in the development of HCC remains unclear. Delineating the effect of ME-CIN on HCC development should help in identifying measures to combat HCC. In this study, we used ME-CIN model mice haploinsufficient in Shugoshin 1 (Sgo1(-/+)) to assess the role of ME-CIN in HCC development. Treatment with the carcinogen azoxymethane caused Sgo1(-/+) ME-CIN model mice to develop HCCs within 6 months, whereas control mice developed no HCC (P < 0.003). The HCC development was associated with expression of early HCC markers (glutamine synthetase, glypican 3, heat shock protein 70, and the serum marker alpha fetoprotein), although without fibrosis. ME-CIN preceded the expression of HCC markers, suggesting that ME-CIN is an important early event in HCC development. In 12-month-old untreated Sgo1 mice, persistent DNA damage, altered gene expression, and spontaneous HCCs were observed. Sgo1 protein accumulated in response to DNA damage in vitro. Overall, Sgo1(-/+)-mediated ME-CIN strongly promoted/progressed development of HCC in the presence of an initiator carcinogen, and it had a mild initiator effect by itself. Use of the ME-CIN model mice should help in identifying drugs to counteract the effects of ME-CIN and should accelerate anti-HCC drug development.

A triple stain of reticulin, glypican-3, and glutamine synthetase: a useful aid in the diagnosis of liver lesions.

CONTEXT: The correct histologic diagnosis of mass lesions of the liver can be difficult, especially in biopsy samples. Reticulin, glypican-3, and glutamine synthetae are stains that can help distinguish hepatocellular carcinoma, hepatic adenoma, and focal nodular hyperplasia. OBJECTIVE: To evaluate the utility of a triple stain of reticulin, glypican-3, and glutamine synthetae in distinguishing hepatocellular carcinoma, hepatic adenoma, and focal nodular hyperplasia. DESIGN: Whole tissue sections and tissue microarrays were evaluated with a triple stain of reticulin, followed by glutamine synthetae (diaminobenzidine, brown chromogen) and glypican-3 (alkaline phosphatase, red chromogen). The 109 cases evaluated included whole tissue section hepatocellular carcinoma (n = 16), tissue microarray hepatocellular carcinoma (n = 19), whole tissue section hepatic adenoma (n = 15), tissue microarray hepatic adenoma (n = 13), whole tissue section focal nodular hyperplasia (n = 13; 12%), tissue microarray focal nodular hyperplasia (n = 13), as well as nonmalignant liver parenchyma adjacent to hepatocellular carcinoma (n = 20). All cases were scored for reticulin being intact or lost, positive or negative staining for glypican-3, and diffuse, maplike, perivenular, or negative staining for glutamine synthetae. RESULTS: The combination of intact reticulin with either glypican-3 negativity or negative glutamine synthetae was 92% sensitive and 95% specific in the distinction of tissue microarray hepatic adenoma from hepatocellular carcinoma. For the distinction of tissue microarray focal nodular hyperplasia and hepatic adenoma, maplike glutamine synthetae was most useful and was 85% sensitive and 100% specific. CONCLUSIONS: The triple stain of reticulin, glypican-3, and glutamine synthetae is useful in the differentiation of hepatocellular carcinoma, hepatic adenoma, and focal nodular hyperplasia on biopsy specimens. Furthermore, this triple stain is advantageous to single stains and can help when aberrant staining patterns are observed.

Relation of glypican-3 and E-cadherin expressions to clinicopathological features and prognosis of mucinous and non-mucinous colorectal adenocarcinoma.

Glypican-3 (GPC3) is a member of the membrane-bound heparin sulfate proteoglycans. E-cadherin is an adhesive receptor that is believed to act as a tumor suppressor gene. Many studies had investigated E-cadherin expressions in colorectal carcinoma (CRC) while only one study had investigated GPC3 expression in CRC. This study aims to investigate expression of GCP3 and E-cadherin in colorectal mucinous carcinoma (MA) and non-mucinous adenocarcinoma (NMA) using manual tissue microarray technique. Tumor tissue specimens are collected from 75 cases of MC and 75 cases of NMA who underwent radical surgery from Jan 2007 to Jan 2012 at the Gastroenterology Centre, Mansoura University, Egypt. Their clinicopathological parameters and survival data were revised and analyzed using established statistical methodologies. High-density manual tissue microarrays were constructed using modified mechanical pencil tip technique and immunohistochemistry for GPC3 and E-cadherin was done. NMA showed higher expression of GPC3 than MA with no statistically significant relation. NMA showed a significantly higher E-cadherin expression than MA. GPC3 and E-cadherin positivity rates were significantly interrelated in NMA, but not in MA, group. In NMA group, there was no significant relation between either GPC3 or E-cadherin expression and the clinicopathological features. In a univariate analysis, neither GPC3 nor E-cadherin expression showed a significant impact on disease-free survival (DFS) or overall survival (OS). GPC3 and E-cadherin expressions are not independent prognostic factors in CRC. However, expressions of both are significantly interrelated in NMA patients, suggesting an excellent interplay between both, in contrast to MA. Further molecular studies are needed to further explore the relationship between GCP3 and E-cadherin in colorectal carcinogenesis.

Expression of glypican 3 enriches hepatocellular carcinoma development-related genes and associates with carcinogenesis in cirrhotic livers.

Glypican-3 (GPC3) protein expression was determined by immunohistochemical analysis from 29 normal livers, 80 cirrhotic livers sample taken near hepatocellular carcinoma (HCC), and 87 cirrhotic livers without HCC. The levels for miR-657 and HCC-related gene mRNAs were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Also, a published microarray dataset was used for gene set enrichment analysis (GSEA) to investigate the relationship between GPC3- and HCC-related gene signatures. Kaplan-Meier analysis was used to evaluate the relationship between GPC3 and HCC recurrence. GPC3 protein expression was not detected in any of the 29 (0%) normal livers, but was detected in 32 of 87 (37%) cirrhotic livers without HCC, and 51 of 80 (64%) cirrhotic liver samples taken near HCC sites (P < 0.001). The GPC3-positive rate in cirrhotic livers of viral origin was 68% (27/40), which was significantly higher than for non-viral cirrhotic livers (11%, 5/47) (P < 0.001). Also, GPC3 expression positively correlated with mRNA expression of HCC-related genes in the qRT-PCR and GSEA evaluations. Furthermore, HCC recurrence in cirrhotic liver samples taken near HCC sites was significantly higher in the GPC3-positive group than the GPC3-negative group (Log-rank P = 0.02, HR = 3.26; 95% CI = 1.20-10.29). This study demonstrated that highly expression of GPC3 could enrich HCC-related genes' mRNA expression and positive associate with dysplasia in cirrhotic livers. Therefore, GPC3 may serve as a precancerous biomarker in cirrhotic livers.