Eunsil Yu

The application of markers (HSP70 GPC3 and GS) in liver biopsies is useful for detection of hepatocellular carcinoma.

BACKGROUND/AIMS Liver biopsy for hepatocellular carcinoma (HCC) detection is largely restricted to small hepatocellular lesions, which are often morphologically challenging, requiring careful distinction between dysplastic nodules (high-grade) and well-differentiated HCC. METHODS We investigated the diagnostic accuracy of a panel of markers (HSP70 GPC3 and GS), previously tested in resection specimens, in a series of liver biopsies of large regenerative nodules (n=13), low-grade dysplastic nodules (n=21), high-grade dysplastic nodules (n=50), very well-differentiated (VWD) (n=17), well-differentiated (WD-G1) (n=40) and G2-3 (n=35) HCC. RESULTS Almost all cases of large regenerative and low-grade dysplastic nodules did not stain while high-grade dysplastic nodules showed 1 marker (22%) but never 2 or 3. For HCC detection the overall accuracy of marker combination was 60.8% (3 markers) and 78.4% (2 markers) with 100% specificity. When restricted to VWD+WD-G1 HCC the accuracy was 57% (3 markers) and 72.9% (2 markers) with 100% specificity. CONCLUSIONS This panel proved useful to detect well-differentiated HCC in biopsy. Two immunoreactive markers (out of 3) are recommended as the most valuable diagnostic combination for HCC detection. The diagnostic accuracy of the panel could be improved using additional markers, as suggested by studies of expression profiling in other human models.

Genomic portrait of resectable hepatocellular carcinomas: implications of RB1 and FGF19 aberrations for patient stratification.

Hepatic resection is the most curative treatment option for early‐stage hepatocellular carcinoma, but is associated with a high recurrence rate, which exceeds 50% at 5 years after surgery. Understanding the genetic basis of hepatocellular carcinoma at surgically curable stages may enable the identification of new molecular biomarkers that accurately identify patients in need of additional early therapeutic interventions. Whole exome sequencing and copy number analysis was performed on 231 hepatocellular carcinomas (72% with hepatitis B viral infection) that were classified as early‐stage hepatocellular carcinomas, candidates for surgical resection. Recurrent mutations were validated by Sanger sequencing. Unsupervised genomic analyses identified an association between specific genetic aberrations and postoperative clinical outcomes. Recurrent somatic mutations were identified in nine genes, including TP53, CTNNB1, AXIN1, RPS6KA3, and RB1. Recurrent homozygous deletions in FAM123A, RB1, and CDKN2A, and high‐copy amplifications in MYC, RSPO2, CCND1, and FGF19 were detected. Pathway analyses of these genes revealed aberrations in the p53, Wnt, PIK3/Ras, cell cycle, and chromatin remodeling pathways. RB1 mutations were significantly associated with cancer‐specific and recurrence‐free survival after resection (multivariate P = 0.038 and P = 0.012, respectively). FGF19 amplifications, known to activate Wnt signaling, were mutually exclusive with CTNNB1 and AXIN1 mutations, and significantly associated with cirrhosis (P = 0.017). Conclusion: RB1 mutations can be used as a prognostic molecular biomarker for resectable hepatocellular carcinoma. Further study is required to investigate the potential role of FGF19 amplification in driving hepatocarcinogenesis in patients with liver cirrhosis and to investigate the potential of anti‐FGF19 treatment in these patients. (Hepatology 2014;60:1971–1981)

Functional interaction between Wnt3 and Frizzled-7 leads to activation of the Wnt/β-catenin signaling pathway in hepatocellular carcinoma cells

BACKGROUND/AIMS The canonical Wnt signaling is frequently activated in human hepatocellular carcinoma (HCC). We previously demonstrated that upregulation of Frizzled-7 receptor (FZD7) in HCC was associated with nuclear accumulation of wild-type beta-catenin. Here, we investigated Wnt ligand(s) that may activate the Wnt/beta-catenin pathway through FZD7 in HCC cells. METHODS To identify Wnt ligand expression, RT-PCR was performed in HCC cells. To evaluate the function of Wnt3 and FZD7 in HCC, we utilized Wnt3 overexpressing FOCUS HCC cells (FOCUS-Wnt3) and human tumors. RESULTS In hepatitis B virus (HBV)-induced HCC, Wnt3 was upregulated in tumor and peritumoral tissues compared to normal liver and downstream beta-catenin target genes were also increased in these samples. Activation of the Wnt/beta-catenin pathway in FOCUS-Wnt3 cells was demonstrated by beta-catenin accumulation, enhanced TCF transcriptional activity and proliferation rate. The activation of Wnt/beta-catenin signaling in FOCUS-Wnt3 was abolished by a knockdown of FZD7 expression by siRNA. More important, a specific Wnt3-FZD7 interaction was observed by co-immunoprecipitation experiments, which suggest that the action of Wnt3 was mediated via FZD7. CONCLUSIONS These findings demonstrate a functional interaction between Wnt3 and FZD7 leading to activation of the Wnt/beta-catenin signaling pathway in HCC cells and may play a role during hepatocarcinogenesis.

Diagnosis of hepatic steatosis and fibrosis by transient elastography in asymptomatic healthy individuals: a prospective study of living related potential liver donors

BackgroundThis prospective study aimed to assess the ability of transient elastography to identify histologic parameters, including steatosis, in asymptomatic healthy individuals such as potential liver donors, and to compare these findings with results in liver disease patients.MethodsForty-seven patients with abnormal liver function and/or hepatitis symptoms and 80 living related potential liver donors were consecutively enrolled, and liver biopsy and a Fibroscan test were performed in each subject. Histologic parameters were evaluated according to METAVIR scale by a single pathologist.ResultsIn liver disease patients, stiffness was significantly correlated with fibrosis stage (Spearman correlation coefficient, 0.700; P < 0.001), and the optimal stiffness cutoff values for F ≥ 2, F ≥ 3, and F = 4 were 7.35, 8.85, and 15.1 kPa respectively. In potential liver donors, however, stiffness was not correlated with fibrosis (0.023; P = 0.851). In the latter group, the area under the receiver-operating characteristics curve was 0.70 (95% confidence interval, 0.58–0.81), and the optimal stiffness cutoff value was 4.00 for F ≥ 2, which was lower than that in liver disease patients. Steatosis was not correlated with stiffness (0.088; P = 0.463) in potential liver donors.ConclusionsTransient elastography has limited value for detecting steatosis in asymptomatic healthy individuals, and the cutoff value for fibrosis should be reevaluated in these subjects.

Ischemic bile duct injury as a serious complication after transarterial chemoembolization in patients with hepatocellular carcinoma.

Background Bile duct injuries after transarterial chemoembolization (TACE) have been reported; however, the exact pathogenic mechanisms and clinical implications of the injuries remain to be clarified. Study A total of 950 consecutive patients with hepatocellular carcinoma (HCC) were studied. Among them, 807 were treated with TACE and the remaining 143 were treated with transarterial chemoinfusion (TACI) of cisplatin. Results None of 143 patients with HCC treated with TACI were found to have any radiographic evidence of biliary injury. In contrast, of the 807 patients treated with TACE, 17 (2%) developed biliary complications. Of all complications, 12 (71%) were subcapsular bilomas; 3 (17%), focal strictures of the common hepatic duct or common bile duct; and 2 (12%), diffuse mild dilatation of the intrahepatic bile ducts. Interestingly, 2 of the 12 bilomas were found in the lobe that was not embolized with gelatin sponge particles. The median numbers of TACE tended to be greater in the patients with focal stricture than in those with bilomas (6.0 vs. 2.5;p = 0.08). All 3 patients with focal strictures and 4 of the 12 patients with bilomas had associated serious bacterial infections at presentation. Conclusions Bilomas seem to be caused by iodized oil rather than gelatin sponge particles; focal strictures of large bile ducts seem to be caused by gelatin sponge particles. We suggest that adjustments in the amounts of iodized oil or gelatin sponge particles and in the sites of embolization may reduce ischemic biliary injuries after TACE.

Application of a new histological staging and grading system for primary biliary cirrhosis to liver biopsy specimens: Interobserver agreement

Recently the authors proposed a new staging and grading system for primary biliary cirrhosis (PBC) that takes into account necroinflammatory activity and histological heterogeneity. Herein is proposed a convenient version of this system. Scores for fibrosis, bile duct loss, and chronic cholestasis were combined for staging: stage 1, total score of 0; stage 2, score 1–3; stage 3, score 4–6; and stage 4, score 7–9. Cholangitis activity (CA) and hepatitis activity (HA) were graded as CA0–3, and HA0–3, respectively. Analysis of interobserver agreement was then conducted. Digital images of 62 needle liver biopsy specimens of PBC were recorded as virtual slides on DVDs that were sent to 28 pathologists, including five located overseas. All participants were able to apply this version in all 62 cases. For staging, kappa was 0.385 (fair agreement) and the concordance rate was 63.9%. For necroinflammatory activity, the kappa and concordance rate were 0.110 (slight agreement) and 36.9% for CA, and 0.197 (slight agreement) and 47% for HA, respectively. In conclusion, this new staging and grading system for PBC seems to be more convenient and practical than those used at present, but more instruction and guidance are recommended for the grading of necroinflammatory activity in practice.

The Expression of Phospho-AKT, Phospho-mTOR, and PTEN in Extrahepatic Cholangiocarcinoma

Purpose: The protein kinase B (AKT) pathway plays a key role in the regulation of cellular survival, apoptosis, and protein translation, and has been shown to have prognostic significance in a number of cancers. We sought to define its role in extrahepatic cholangiocarcinoma. Experimental Design: Two hundred twenty-one extrahepatic cholangiocarcinoma patients with clinicopathologic data, including survival, were arrayed into tissue microarrays. Phosphorylated AKT (p-AKT), phosphorylated mammalian target of rapamycin (p-mTOR), and total phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein expressions were studied with multiplex tissue immunoblotting assay. Results: Expressions of p-AKT and p-mTOR were significantly increased in extrahepatic cholangiocarcinoma cases compared with normal and dysplastic bile duct epithelium (P < 0.05 both). Decreased PTEN expression was observed in patients with increasing depth of invasion (P < 0.05), T classification (P < 0.05), and stage grouping (P < 0.05), and the presence of invasion of the pancreas (P < 0.05) and duodenum (P < 0.05). Decreased PTEN expression (P = 0.004) as well as decreased PTEN/p-AKT (P = 0.003) and PTEN/p-mTOR (P = 0.009) expression showed shorter survival by univariate but not by multivariate analysis. Conclusions: The AKT pathway is activated in a subset of extrahepatic cholangiocarcinoma. Elevated PTEN expression correlates with longer survival. Quantitative data obtained by multiplex tissue immunoblotting may provide additional information than assessment of immunohistochemistry alone. Quantitative analysis of PTEN, PTEN/p-AKT and PTEN/p-mTOR shows differences in survival by univariate analysis.

Metastatic tumor antigen 1 is closely associated with frequent postoperative recurrence and poor survival in patients with hepatocellular carcinoma

Metastatic tumor antigen 1 (MTA1) is known to play a role in angiogenic processes as a stabilizer of hypoxia‐inducible factor 1‐α (HIF1‐α). In this study, we examined whether overexpression of MTA1 affects the recurrence of hepatocellular carcinoma (HCC) after surgical resection and the survival of the patients. A total of 506 HCC patients who underwent hepatic resection were included in the study. They were followed up for a median of 43 months (range, 1‐96 months) after hepatectomy. MTA1 expression levels were determined by the proportion of immunopositive cells (none, all negative; +, <50%; ++, >50%). The relationships between MTA1 expression and the HCC histological features, the appearance of recurrent HCC after surgical resection, and the survival of the patients were examined. Eighty‐eight cases (17%) of the HCCs were positive for MTA1, although the surrounding liver tissues were all negative for MTA1; 62 cases were + and 26 cases were ++. Increased MTA1 expression levels in HCC were correlated with larger tumors (P = 0.04), perinodal extension (P = 0.03), and microvascular invasion (P = 0.008). Histological differentiation had marginal significance (P = 0.056). However, there was no association between MTA1 expression and age, sex, Child‐Pugh class, and capsule invasion of HCC. Interestingly, MTA1 expression levels were significantly greater in hepatitis B virus (HBV)‐associated HCC compared with hepatitis C virus (HCV)‐associated HCC (P = 0.017). The cumulative recurrence rates of MTA1‐positive HCCs were markedly greater than those of MTA1‐negative HCCs (P < 0.0001). The cumulative survival rates of patients with MTA1‐positive HCCs were significantly shorter than those of patients with MTA1‐negative HCCs (P < 0.0001). In conclusion, our data indicate that MTA1 is closely associated with microvascular invasion, frequent postoperative recurrence, and poor survival of HCC patients, especially in those with HBV‐associated HCC. (HEPATOLOGY 2008;47:929–936.)

p16 Hypermethylation in the early stage of hepatitis B virus- associated hepatocarcinogenesis

Abnormality of the p16 expression is involved in the pathogenesis of hepatocellular carcinoma (HCC), and hypermethylation of p16 gene is known as a major p16 inactivation mechanism. Cirrhotic nodule (CN) is now regarded as a preneoplastic lesion that is frequently associated with microscopic foci of HCC through dysplastic nodules (DNs). This observation clearly supports a multistep hepatocarcinogenesis from CNs through DNs. We thus examined the methylation status of p16 gene in HCCs surrounded by DNs and CNs to define the significance of p16 hypermethylation in the early stage of hepatocarcinogenesis. We tested 24 hepatitis B virus (HBV)-associated CNs, 37 DNs, and 18 HCCs within DNs that were microdissected from paraffin-embedded tissue sections. Frequency of p16 hypermethylation was significantly high in HCCs within DNs (15/18. 83.3%) and it increased from CNs (15/24. 62.5%) through DNs (26/37, 70.3%). Interestingly, 11 out of 12 (91.7%) HCC associated with methylation-positive DNs revealed hypermethylation of p16, and 18 out of 23 (78.2%) DNs associated with methylation-positive CNs showed p16 hypermethylation. These data suggest that p16 hypermethylation in the early stages, CNs and DNs may predispose to HCC. In addition, p16 methylation status of five cell lines with or without HBV infection was examined to test whether the high frequency of hypermethylation is related to HBV infection. HBV-infected cell lines were exclusively methylation-positive. These data suggest that high frequency of hypermethylation may be associated with hepatitis B virus infection.

Septated cystic tumors of the pancreas: is it possible to treat them by endoscopic ultrasonography-guided intervention?

Objectives. Endoscopic ultrasonography (EUS)-guided intervention has recently been tried in the treatment of cystic tumors of the pancreas. Factors that can influence the treatment response include cyst wall thickness and the presence of septation and mural nodules. This study aimed to evaluate the effectiveness of EUS-guided ethanol lavage with paclitaxel injection (EUS-EP) in the treatment of septated cystic tumors of the pancreas. Methods. Ten patients with oligolocular septated cystic tumors underwent EUS-EP and the treatment response was analyzed by measuring cyst volume before and more than 6 months after treatment. Results. The 10 patients comprised 7 women and 3 men, of mean age 38.4 years (range 22–54 years). Median tumor diameter was 29.5 mm (range 20–68 mm), while median tumor volume was 5.07 ml (range 1.51–68.74 ml). Median carcinoembryonic antigen and amylase concentrations in the cyst fluid were 39.2 ng/ml (range 1–8,190 ng/ml) and 115.5 U/L (range 5–75,633 U/L), respectively. Three tumors were diagnosed as mucinous cystic neoplasms, four as serous cystadenomas, and three as indeterminate cysts. Median tumor volume had decreased to 0.54 ml (range 0.00–12.42 ml). Complete resolution was achieved in six patients and partial resolution in two, whereas two patients had persistent cysts. Two patients with persistent cysts underwent surgical resection, and focal remnant neoplastic epithelial lining was observed on the resected specimens. Conclusions. EUS-EP resulted in complete resolution in 6 of 10 patients with septated cystic tumors. Careful patient selection and tailored intervention are required to maximize the therapeutic efficacy of this procedure.