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Radioiodine whole body scan (WBS), related to sodium iodide symporter (NIS) function, is widely used to detect recurrence/metastasis in postoperative patients with thyroid cancer. However, the normal thymic uptake of radioiodine has occasionally been observed in young patients. We evaluated the expression of thyroid-related genes and proteins in the human thymus. Thymic tissues were obtained from 22 patients with thyroid cancer patients of all ages. The expression of NIS, thyroid-stimulating hormone receptor (TSHR), thyroperoxidase (TPO), and thyroglobulin (Tg) was investigated using immunohistochemistry and quantitative RT-PCR. NIS and TSHR were expressed in 18 (81.8%) and 19 samples (86.4%), respectively, whereas TPO was expressed in five samples (22.7%). Three thyroid-related proteins were localized to Hassall’s corpuscles and thymocytes. In contrast, Tg was detected in a single patient (4.5%) localized to vascular endothelial cells. The expression of thyroid-related proteins was not increased in young thymic tissues compared to that in old thymic tissues. In conclusion, the expression of NIS and TSHR was detected in the majority of normal thymus samples, whereas that of TPO was detected less frequently, and that of Tg was detected rarely. The increased thymic uptake of radioiodine in young patients is not due to the increased expression of NIS.

Background The Hippo pathway plays a vital role in liver regeneration and development by determining cellular lineage and regulating cell proliferation and apoptosis. In this study, we aimed to assess the role of the Hippo pathway in hepatic carcinogenesis and morphogenesis by examining Yes-associated protein 1 (YAP1) expression in the spectrum of hepatic carcinomas based on cellular lineage. Methods We examined 913 primary hepatic carcinomas, including hepatocellular carcinomas (HCCs), combined hepatocellular and cholangiocarcinomas (cHC-CCAs), intrahepatic cholangiocarcinomas (IHCCAs) and perihilar extrahepatic bile duct carcinomas (EHBCAs). Our study group was categorized into 8 disease groups, based on histological diagnosis and cytokeratin 19 (CK19) expression, and immunohistochemistry was used to detect and compare YAP1 expression levels between the groups. The eight disease groups we identified were: 1) CK19(−) HCC, 2) CK19(−) scirrhous HCC, 3) CK19(+) HCC, 4) stem cell feature of cHC-CCA, 5) classical cHC-CCA, 6) cholangiolocellular IHCCA, 7) non-cholangiolocellular IHCCA, and 8) EHBCA. Results Positive rates of YAP1 were the highest in the EHBCA group (21%). CK19(+) HCC and non-cholangiolocellular IHCCA groups also showed high expression levels (10% -11%), while the CK19 (−) HCC, CK19 (−) scirrhous HCC, cHC-CCA, and cholangiolocellular IHCCA groups showed low expression levels, ranging between 0% and 5%. Survival analysis, restricted to pT1 stage HCCs and IHCCAs, showed poor overall survival for YAP1(+) IHCCA patients (39 ± 17 vs. 109 ± 10 months, mean ± SD, log rank p-value 0.005). For HCCs, a trend of poor progression-free survival for YAP1(+) HCCs was observed (39 ± 18 vs. 81 ± 5 months, mean ± SD, log rank p-value 0.205) Conclusions YAP1 activation was more commonly found in CCAs than in pure HCCs. However, a differing pattern of YAP1 expression between cHC-CCAs and CK19(+) HCCs and the poor prognosis of YAP1 positive hepatic carcinomas suggests that YAP1 may have a preferential role in aggressive tumor behavior, rather than in the determination of cellular lineage in hepatic carcinomas. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3431-1) contains supplementary material, which is available to authorized users.

Spleen tyrosine kinase (SYK) has predominantly been studied in hematopoietic cells, where it is involved in immunoreceptor-mediated signaling. However, SYK expression has been shown in numerous non-hematopoietic cells, and its downregulation has been shown to be involved in tumor formation and progression. SYK methylation has been demonstrated to identify a subset of hepatocellular carcinoma (HCC) cases with poor prognosis, but little is known regarding the biological role of SYK in HCC. We found that SYK methylation is a common event in HCC, and is inversely associated with its expression. We established stable HCC cell lines with inducible SYK expression vectors, and compared the differential RNA expression profiles of HCC cell lines with or without the induction of SYK. Gene ontology analysis revealed that the SYK-regulated genes were enriched for genes involved in cell adhesion. Accordingly, we found that the induction of SYK expression increased the adhesion of cells to fibronectin and decreased cell migration and invasion, and that cessation of SYK overexpression increased cell migration and invasion. Our findings suggest that SYK is involved in regulating cell to matrix adhesions, and that SYK loss affects the migration, and invasion of HCC cells.

Aberrant regulation of histone deacetylase 2 (HDAC2) contributes to malignant progression in various cancers, but the underlying mechanism leading to the activation of oncogenic HDAC2 remains unknown. In this study, we show that HDAC2 expression is upregulated in a large cohort of patients with human hepatocellular carcinoma, and that high expression of HDAC2 was significantly associated with poor prognosis of patients with hepatocellular carcinoma. We found that mTORC1/NF-κBp50 signaling is necessary for the growth factor–induced HDAC2 and is sustained in hepatocellular carcinoma, but not in normal hepatic cells. Growth factor–induced mTORC1 activates the nuclear translocation of NF-κBp50, where it binds to the intragenic sequences of the HDAC2 gene and promotes its transcription. Hepatocellular carcinoma tissues derived from chemical-induced mouse and rat liver cancer models validated that mTORC1 activation and NF-κBp50 nuclear translocation are essential for the transcriptional activation of oncogenic HDAC2 in hepatocellular carcinoma. In addition, we demonstrate that HDAC2 is required to maintain mTORC1 activity by stabilizing the mTOR/RAPTOR complex. Elevated expression of HDAC2 triggers a positive feedback loop that activates AKT phosphorylation via the transcriptional modulation of phosphoinositide signaling molecules. Bioinformatics analysis of HDAC2 signature and immunoblot analysis of mesenchymal genes also evidenced that HDAC2 plays a role in the malignant behavior of tumor cells by Snail induction and simultaneously E-cadherin suppression in hepatocellular carcinoma cells. These findings establish a molecular mechanism responsible for the activation of oncogenic HDAC2, which explains how growth factor–induced HDAC2 maintains mitogenic signaling and function during hepatocellular malignant progression and provide a novel strategy for therapeutic intervention in liver cancer. Cancer Res; 74(6); 1728–38. ©2014 AACR.

Ethnopharmacology relevance Dendropanax morbifera Leveille (DM) has been used in traditional medicines for infectious and skin diseases, and dysmenorrhea. It exhibits a diverse therapeutic potential including anti-cancer, anti-thrombotic, anti-diabetic, anti-oxidant, and anti-inflammatory activities. Aim of the study Despite promising health benefits of DM, knowledge of its potential adverse effects is very limited. The current study focused on the investigation of subchronic toxicity and genotoxicity of extract obtained from DM according to the test guidelines published by the Organization for Economic Cooperation and Development. Materials and methods We conducted a toxicological evaluation of DM extracts using 14-day repeated-dose toxicity study and 13-week repeated-dose toxicity study in Sprague-Dawley rats administered orally at doses of 500, 1000, or 2000 mg/kg/day. The clastogenicity of DM extract was also evaluated by in vitro chromosome aberration assay and in vivo micronucleus assay. Results Assessment of subchronic toxicity of DM extract by oral administration in rats revealed unremarkable treatment-related findings with respect to food/water consumption, body weight, mortality, urinalysis, hematology, serum biochemistry, necropsy, organ weight and histopathology at doses of 500, 1000, and 2000 mg/kg. Accordingly, the level of no-observed-adverse-effect for DM extract in 13-week subchronic toxicity study was considered to be 2000 mg/kg/day in rats. The data observed from in vitro chromosome aberration assay and in vivo micronucleus assay exclude any clastogenicity of DM extract. Conclusion The results suggest that the oral consumption of DM extract has no adverse effects in humans and represents a safe traditional medicine.

Background & Aims Hepatocellular carcinomas (HCCs) expressing "stemness"-related markers have been associated with aggressive biological behavior and poor prognosis. We examined the relationship between "stemness"-related protein expression and telomere length, hTERT and shelterin complex protein expression and chromosomal instability. Methods Quantitative fluorescent in situ hybridization for telomere length, immunohistochemistry for K19, EpCAM, CD133, c-kit, HepPar1, hTERT, TRF1, TRF2, POT1, RAP1 and TPP1, and TUNEL assay were performed in 137 HCCs, and array comparative genomic hybridization was performed with 24 HCCs. Results Telomeres were significantly longer in HCCs expressing "stemness"-related proteins (K19: p p =0.002, CD133: p =0.002). On analyzing different tumor cells within EpCAM-expressing HCCs, EpCAM-positive tumor cells showed longer telomeres (1.329±0.246) compared to EpCAM-negative tumor cells (0.996±0.381) within the same HCCs ( p =0.031). Telomeres were significantly longer in HCCs expressing hTERT ( p =0.048) and RAP1 proteins ( p =0.031). K19-expressing HCCs expressed hTERT ( p =0.002), TRF2 ( p =0.001) and TPP1 ( p =0.013) more frequently compared to K19-negative HCCs. EpCAM-positivity was associated with more frequent hTERT ( p =0.028), TPP1 ( p =0.017), TRF2 ( p =0.027) and POT1 ( p =0.004) expression. Copy number alterations were more frequent in K19 and EpCAM-expressing HCCs compared to HCCs without these markers (K19: p =0.038, EpCAM: p =0.009). HCCs with longer telomeres were associated with a shorter overall ( p =0.019) and disease-free survivals ( p =0.049), and decreased disease-free survivals were seen in TRF2-positive HCCs ( p =0.018). Conclusions HCCs expressing "stemness"-related proteins are characterized by increased telomere length, increased expression of hTERT and shelterin complex proteins, and increased chromosomal instability compared to conventional HCCs. Longer telomeres and TRF2 expression in HCCs are associated with poor patient outcomes.

The capability to engraft into degenerative environment or damaged tissues is considered crucial to maximize the therapeutic effect of stem cells in tissue regeneration. Human chorionic plate-derived mesenchymal stem cells (CP-MSCs) isolated from the placenta have been reported to have therapeutic effects in animal models of liver injury. However, the effect of transplantation route has not been evaluated. Thus, we investigated to identify optimal transplantation conditions of CP-MSCs for functional recovery of injured liver. PKH26 labeled CP-MSCs was engrafted into carbon tetrachloride (CCl4)-injured rat model through direct transplantation into the liver (DTP), intrasplenic transplantation (STP), and intravenous transplantation via the tail vein (TTP). Non transplanted (NTP) rats were maintained as sham controls. Serum and liver tissues were analyzed post 1-, 2-, 3-week after transplantation. The engraftment of cells was higher in DTP and STP group until 3-week post transplantation. In blood chemistry, the levels of glutamate-oxaloacetate transaminase (GOT/AST), glutamate-pyruvate transaminase (GPT/ALT) and total bilirubin (TBIL) in DTP and STP rats were significantly decreased compare to those of NTP rats (p < 0.01). In addition, the expression and deposition of type I collagen in DTP and STP rats was significantly reduced when they compared with NTP animals (p < 0.01). Therapeutic efficacy was better in DTP and STP rats than in TTP group. These results suggest that the administration of CP-MSCs via STP is an effective way for their therapeutic potential. These results provide useful guidelines for the application of basic transplantation technology to the cell therapy of liver disease using placenta-derived stem cells.

Umbilical cord-derived mesenchymal stem cells (UC-MSCs) therapy might be an alternative to liver transplantation for acute or chronic liver injury. The aim of this study was to evaluate the efficacy of human UC-MSCs on carbon tetrachloride (CCl4)-induced acute liver injury. In addition, its toxicity, tumorigenicity, and biodistribution were determined. Significant hepatoprotective effects of hUC-MSCs with decreased levels of hepatocellular necrosis and lobular neutrophilic infiltration were found. Regarding the safety of hUC-MSCs, no serious hUC-MSCs-related changes (body weight, food/water consumption, clinical symptom, urinalysis, hematology, clinical chemistry, organ weight, and histopathology) were observed in a 13-week subchronic toxicity study. In a 26-week tumorigenicity study, no mice developed tumor related to hUC-MSCs transplantation up to 1 × 108 cells/kg. In particular, human mitochondrial sequence detection revealed that most hUC-MSCs were cleared from the major organs of the mice at 13 weeks after transplantation. There was no systemic toxicity or neoplastic finding either. Taken together, these results suggested that hUC-MSCs have great potential for future clinical treatment of acute liver disease.

We compared the microscopic and nuclear morphometric characteristics of classical chromophobe renal cell carcinoma (C-ChRC) and renal oncocytoma (RO) and applied meaningful characteristics to differentiate eosinophilic chromophobe renal cell carcinoma (E-ChRC) from RO that has overlapping histology (RO-OH) with E-ChRC to know the usefulness of nuclear morphometry. Microscopic and morphometric characteristics were evaluated in 24 C-ChRCs, 6 E-ChRCs, 5 RO-OHs, and 25 classical ROs (C-ROs). The microscopic findings favoring C-ChRC were rasinoid nuclei, perinuclear halo, and distinct cytoplasmic membrane. Characteristic for C-RO was either stromal edema or hyalinization. The morphometric values of nearest nuclear distance, shortest nuclear diameter, and nuclear diameter ratio were significantly different between C-ChRC and C-RO. However, it was impossible to distinguish E-ChRC from RO-OH by histology and nuclear morphometry. The results of our study show that nuclear morphometry and histomorphology can distinguish between C-ChRC and C-RO but not between E-ChRC and RO-OH.

Although increased evidence has suggested that epithelial-mesenchymal transition has been implicated in cancer invasion and is associated with poor prognosis, its significance in cholangiocarcinoma remains unclear. We evaluated the levels of expression of epithelial-mesenchymal transition-related genes and proteins in 2 established human cholangiocarcinoma cell lines with different morphological characteristics and performed transwell cell invasion assays. Furthermore, we investigated the association between altered expression of 6 epithelial-mesenchymal transition-related proteins and clinical outcomes in human cholangiocarcinoma patients (n = 119) by immunohistochemistry using a tissue microarray approach. Comparative analysis of protein and messenger RNA expression revealed that the cell line with less differentiation (JCK) showed increased expression of mesenchymal markers and zinc-finger proteins and decreased expression of epithelial markers. The invasion activity of JCK cells was significantly higher than that of cells from OZ cell lines. Tissue microarray analysis revealed that the combined expression pattern of 6 epithelial-mesenchymal transition-related proteins predicted shortened disease-free survival (13.0 versus 22.0 months, P = .033) and overall survival (23.0 versus 63.0 months, P = .003) and was confirmed as an independent unfavorable prognostic factor for survival in multivariate survival analysis (disease-free survival, P = .028 for the 3 epithelial-mesenchymal transition-related markers; overall survival, P = .010 for the 6 epithelial-mesenchymal transition-related markers). In conclusion, our results suggest that altered expression of a number of epithelial-mesenchymal transition-related genes in tumor cells with poor differentiation may explain their increased invasive ability. Our results also suggest that altered expression of a suite of epithelial-mesenchymal transition-related proteins could be used as a tool to predict poor outcomes in human cholangiocarcinoma patients.

Hepatitis B virus (HBV) is the most common cause of chronic liver disease in South Korea and is one of the major risk factors of hepatocellular carcinoma (HCC) worldwide. Although various radiologic techniques for early detection and medical or surgical methods have successfully controlled the primary tumor, HCC still shows unsatisfactory long-term prognosis after surgical resection probably due to high recurrence rate.1 Most studies reported greater than 70% recurrence rate within a 5-year period. This recurrence, rather than the underlying cirrhosis, is the major cause of death.2-4 Hence, many investigations have studied risk factors for HCC recurrence after curative resection. Clinicopathologic characteristics of HCC such as micrometastasis, vascular invasion, and positive resection margin are well-known risk factors for tumor recurrence.5 Recently, peritumoral non-neoplastic liver parenchyma (PNLP) has been suggested to be an important predictive marker of HCC recurrence after surgical resection.6-8 PNLP can be defined as hepatic parenchyma without histologic evidence of tumors. However, in patients with chronic liver disease, PNLP has two different levels; the first is adjacent parenchyma that is affected by tumors and the second one is remote parenchyma that is not affected by primary tumors. Adjacent PNLP is claimed to be influenced by mass effect of tumor proper and several tumor-producing factors via paracrine or autocrine manners, indirectly depicting the trait of tumors. Meanwhile, remote PNLP may express the patient's underlying chronic liver disease and indirectly the trait of tumorigenesis, implying new occurrence of HCCs. It is therefore important to determine the exact distance of adjacent PNLP that is directly influenced by the tumor, which differs from remote PNLP, the background liver parenchyma. However, published reports lack consistency on the precise distance that defines adjacent PNLP. Some authors used the farthest non-cancerous liver parenchyma in the entire surgical specimen.9 However, there were some other studies that have limited specimens to a distance of 10 mm from the tumor,10 while others have simply made use of the peritumoral liver tissue that was available at the time of the study.11 In this study, we sought the pathologic characteristics of PNLP of HCCs in chronic HBV patients in order to suggest a reasonable cut-off point between adjacent and remote PNLP from the histologic view point.

Extrahepatic cholangiocarcinoma (EHC) is usually difficult to diagnose by bile cytology because of cellular disintegration. However, DNA samples from bile fluid can provide sufficient materials to screen for the presence of EHC. We developed DNA methylation marker panels that can be used for MethyLight assay-based detection of EHC in bile fluid specimens. The methylation status of 59 DNA methylation markers was investigated in 20 EHC and 20 non-neoplastic gallbladder tissue samples with MethyLight assay to determine cancer-specific DNA methylation markers. Through assaying cancer-specific DNA methylation markers in a training set (n = 40) and validation set (n = 45) of bile fluid specimens from patients with EHC or those without cancer, we selected suitable marker panels that were assessed for their performance in a third set (test set; n = 40). Four marker panels showed a sensitivity of 60% or more and a specificity of 100% in both the training and validation sets, whereas bile cytology displayed a sensitivity of 40% to 46% and a specificity of 100%. In an independent test set of bile fluid samples, a five-gene panel (CCND2, CDH13, GRIN2B, RUNX3, and TWIST1) detected EHC at a sensitivity of 83%, which was far higher than that of bile cytology (46%, P = 0.004). Using bile fluids, a methylation assay consisting of a five-gene panel may be useful for detecting EHC and in helping to increase the sensitivity of preoperative diagnoses.

To retrospectively assess feasibility of dual-source dual-energy (DSDE) computed tomography (CT) for evaluation of hepatic iron accumulation in a liver phantom and liver transplantation candidates and to compare its accuracy with that of 3-T magnetic resonance (MR) imaging.This study was approved by the institutional review board; informed consent was waived. A liver agar phantom containing six tubes of iron (concentrations, 100-5000 mg of iron per 100 mL of solution) was scanned at 80 and 140 kVp with both DSDE mode and single-source dual-energy (SSDE) CT with sequential scanning mode. Difference of averaged attenuation between 80 and 140 kVp at CT (ΔH) was measured and correlated with iron concentration. Thirty-two liver transplant recipients and 55 donors who underwent DSDE CT at 80 and 140 kVp were included. Twenty-three underwent 3-T liver MR with dual-echo in-phase and opposed-phase T1-weighted and spin-echo T2-weighted imaging. Hepatic ΔH was measured at CT. On T1- and T2-weighted MR images, iron indexes were calculated. Degree of iron accumulation and macrosteatosis were determined at histologic examination (reference). Diagnostic performance of ΔH at CT and iron indexes at MR for diagnosing clinically important iron accumulation was evaluated (receiver operating characteristic [ROC] analysis).For phantom study, ΔH obtained from both DSDE mode and SSDE CT with sequential scanning mode was correlated with iron concentration (correlation coefficient, 1.00 and 0.943, respectively; P = .173). ΔH (13.53) in 10 patients with clinically important (≥ 10%) iron accumulation was significantly higher than that (7.39) in 77 patients with normal or mild iron deposition (P.001). ΔH was significantly correlated with degree of iron accumulation (correlation coefficient, 0.430; P.001) but not with degree of hepatic macrosteatosis (P = .216). Area under the ROC curve for diagnosing clinically important iron accumulation was 0.881 and 0.897 with CT and MR, respectively (P = .851).DSDE CT is accurate for diagnosing clinically important hepatic iron accumulation without confounding influence of hepatic steatosis, with diagnostic performance on par with MR.

Shin E, Kim S-H, Jeong H-Y, Jang J-J, Lee K. Nuclear expression of S-phase kinase–associated protein 2 predicts poor prognosis of hepatocellular carcinoma. APMIS 2012; 120: 349–57. The S-phase kinase–associated protein 2 (Skp2), which ubiquitinates the cell cycle inhibitor p27Kip1 and targets it for degradation, is commonly overexpressed in human cancers and is associated with poor prognosis in several cancers. The aim of this study was to investigate Skp2 expression and its clinicopathologic significance in surgically resected hepatocellular carcinomas. We collected 359 hepatocellular carcinoma samples and evaluated Skp2 protein expression in cytoplasmic and nuclear fractions by immunohistochemistry using a tissue microarray method. Among the 359 patients, nuclear expression of Skp2 was observed in 41 (10.38%), and cytoplasmic expression of Skp2 was observed in 195 (49.37%). Of the several clinicopathologic variables examined, high Edmonson-Steiner grade and early recurrence correlated with nuclear expression of Skp2 (p = 0.000 and 0.022, respectively). Cytoplasmic expression of Skp2 correlated negatively with microvascular and macrovascular invasion, tumor size, histologic grade, and overall survival. Multivariate analysis revealed that nuclear expression of Skp2 correlated with short disease-free survival. Our findings suggest that nuclear expression of Skp2 may be used as an independent predictor of poor prognosis for hepatocellular carcinoma, whereas cytoplasmic expression of Skp2 may indicate less aggressive disease.

Background/Aims Cluster differentiation 44 standard isoform (CD44s) is a transmembrane glycoprotein. CD44s is a known prognostic factor in various cancers, due to its involvement in tumor cell growth, invasion and metastasis. Its prognostic role, however, is debated because it can be a positive or negative prognostic factor depending on tumor type and is still an ambiguous prognostic indicator in other cancers, especially hepatocellular carcinoma (HCC). We investigated the relationship between CD44s expression and survival in HCC patients. Methods A total of 260 HCC samples were collected to generate a tissue microarray. Staining of the arrays with a primary mouse CD44s monoclonal antibody was followed by evaluation of the relationship between CD44s expression and tumor differentiation. The effect of CD44s expression on patient survival was analyzed. Results CD44s protein expression correlated with histological grade (most and worst Edmondson grade) of the HCC (p=0.029 and p=0.039, respectively) and adversely affected the disease free survival period based on univariate and multivariate analyses (p=0.038 and p=0.077, respectively). Conclusions High CD44s protein expression correlates with shorter disease free survival and poorly differentiated HCC. CD44s-targeted therapy may be efficacious for HCC treatment in the future.

The hypoxic condition within large or infiltrative hypovascular tumors produces intracellular acidification, which could activate many signaling pathways and augment cancer cell growth and invasion. Carbonic anhydrase-IX (CA-IX) is an enzyme lowering pH. This study is to examine whether hypoxia induces CA-IX in hepatocellular carcinoma (HCC) cells, and to evaluate its clinical implication in HCC patients. Human HCC cell lines (Huh-7 and HepG2 cells) were used, and cell growth was assessed using MTS assay. CA-IX expression and apoptotic/kinase signaling were evaluated using immunoblotting. The cells were transfected with CA-IX-specific siRNA, or treated with its inhibitor 4-(2-aminoethyl) benzenesulfonamide (CAI#1), and/or the hexokinase II inhibitor, 3-bromopyruvate (3-BP). A clinic pathological analysis of 69 patients who underwent an HCC resection was performed using a tissue array. Incubation of HCC cells under hypoxia (1% O2, 5% CO2, 94% N2) for 36 h significantly increased CA-IX expression level. CAI#1 (400 μmol/L) or CA-IX siRNA (100 μmol/L) did not influence HCC cell growth and induce apoptosis. However, CAI#1 or CA-IX siRNA at these concentrations enhanced the apoptosis induced by 3-BP (100 μmol/L). This enhancement was attributed to increased ER stress and JNK activation, as compared with 3-BP alone. Furthermore, a clinic pathological analysis of 69 HCC patients revealed that tumor CA-IX intensity was inversely related to E-cadherin intensity. Inhibition of hypoxia-induced CA-IX enhances hexokinase II inhibitor-induced HCC apoptosis. Furthermore, CA-IX expression profiles may have prognostic implications in HCC patients. Thus, the inhibition of CA-IX, in combination with a hexokinase II inhibitor, may be therapeutically useful in patients with HCCs that are aggressively growing in a hypoxic environment.

The tissue environment in the region of hepatocellular carcinoma (HCC) influences both vascular invasion and recurrence. Thus, HCC patient prognosis depends on the characteristics not only of the tumor but also those of adjacent surrounding liver tissue. Expression profiles of both tumor and adjacent liver tissue following curative resection were measured to discriminate 56 hepatitis B virus-positive HCC patients into subgroups based on survival risk. This approach was further tested in 40 patients. Expression profiles of both tumor and adjacent liver tissue successfully discriminated 56 training samples into 2 subgroups, those at low- or high-risk for survival and recurrence. However, the prognostic gene set selected for tumor tissue was quite different from that for adjacent tissues. This variation in prognostic genes resulted in a change in allocation of patients within each low- or high-risk group. Combination of survival subgroups from tumor and adjacent liver tissue significantly improved the prediction of prognostic outcome. This integrative approach was confirmed to be effective in a further 40 test patients. A clinicopathological study showed that survival subgroups divided by tumor and adjacent liver tissue gene expression were also statistically associated with UICC stage and extent of cell differentiation, respectively. Variation in gene expression during the nontumor stage as well as the tumor stage may affect the prognosis of HCC patients, and integration of the gene expression profiles of HCC and adjacent liver tissue increases discriminatory effectiveness between patient groups, predicting clinical outcomes with enhanced statistical reliability.

Heat shock protein 70 (HSP70) and glutamine synthetase (GS) have been proposed to be promising markers for the differentiation of malignant and benign hepatocellular lesions. The aim of this study was to investigate the clinicopathological significance of the expression of HSP70 and GS in surgically resected hepatocellular carcinoma (HCC).The authors collected 412 HCC samples and 120 non-neoplastic hepatic tissue samples and performed an immunohistochemical study.HSP70 staining was observed in 282 of 392 HCC samples (71.9%), and GS immunoreactivity was observed in 212 of 395 HCC cases (53.7%). Of the several clinicopathological parameters examined, microscopic vascular invasion, a large tumor size, and a high Edmonson-Steiner grade were found to be correlated with positive staining for HSP70 (P = 0.032, 0.002, and 0.012, respectively). Survival analysis showed a correlation between HSP70 expression and disease-free survival. GS was not found to be related to clinicopathological parameters.The findings of the present study suggest that HSP70 be viewed as a predictor of prognosis as well as a useful diagnostic marker for HCC.

Goal: In this study, we attempted to evaluate the prognosis of combined hepatocellular and cholangiocarcinoma (cHCC-CC) with comparison to hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CC). Background: The prognosis of cHCC-CC has not been fully elucidated. In this study, we attempted to evaluate the prognosis of cHCC-CC with comparison to HCC and CC. Study: Consecutive patients who underwent curative resection for cHCC-CC at a single tertiary care center in Korea and their age, sex, and Child-Turcotte-Pugh class matched HCC and CC patients were included. We evaluated time-to-recurrence (TTR) and overall survival (OS) of cHCC-CC cases and compared them with HCC and CC patients. Results: Thirty cHCC-CC, 60 HCC, and 60 CC patients were included. For cHCC-CC group, the median TTR and OS were 5.4 and 18.0 months. After adjustment for confounding factors, the cHCC-CC group had a shorter TTR than did HCC group [relative risk (RR), 2.76; P < 0.001] and CC group (RR, 2.00; P = 0.013), and a shorter OS than HCC group (RR, 4.70; P < 0.001). Compared with the each stage I diseases, cHCC-CC had shorter TTR than HCC (RR, 4.59; P = 0.001) and CC (RR, 2.74, P = 0.015) and shorter OS than HCC (RR, 9.35; P = 0.001). Conclusions: The results of this study indicated that cHCC-CC had a significantly poorer prognosis than HCC and CC even after curative resection.

In a previous report we demonstrated that the transcriptomic response of liver tissue was specific to toxicants, and a characteristic molecular signature could be used as an early prognostic biomarker in rats. It is necessary to determine the transcriptomic response to toxicants in peripheral blood for application to the human system. Volatile organic compounds (VOCs) comprise a major group of pollutants which significantly affect the chemistry of the atmosphere and human health. In this study we identified and validated the specific molecular signatures of toxicants in rat whole blood as early predictors of environmental toxicants. VOCs (dichloromethane, ethylbenzene, and trichloroethylene) were administered to 11-week-old SD male rats after 48 h of exposure, peripheral whole blood was subjected to expression profiling analysis. Unsupervised gene expression analysis resulted in a characteristic molecular signature for each toxicant, and supervised analysis identified 1,217 outlier genes as distinct molecular signatures discerning VOC exposure from healthy controls. Further analysis of multi-classification suggested 337 genes as early detective molecular markers for three VOCs with 100% accuracy. A large-scale gene expression analysis of a different VOC exposure animal model suggested that characteristic expression profiles exist in blood cells and multi-classification of this VOC-specific molecular signature can discriminate each toxicant at an early exposure time. This blood expression signature can thus be used as discernable surrogate marker for detection of biological responses to VOC exposure in an environment.

Aim: Hepatic stellate cells (HSCs) have immune regulatory functions. Mesalamine is an effective immune regulatory drug for inflammatory bowel disease. Thus, we hypothesized that mesalamine may also modulate the immune regulatory functions of HSCs. Since B7-H1 plays a crucial role in regulating T-cell apoptosis, we evaluated if mesalamine induces B7-H1 expression on HSCs, and if so, whether mesalamine attenuates autoimmune liver injury in vivo. Methods: LX-2 cells, an immortalized human HSC cell line, and human peripheral T-cells were used in this study. B7-H1 expression on LX-2 cells following mesalamine treatment was examined by using flow cytometry. Cell viability was analyzed by MTS assay. Concanavalin-A (ConA) mice hepatitis model was used for in vivo study. Results: Flow cytometry showed that mesalamine treatment increased the B7-H1-expressing LX-2 cell fraction from 45.4% to 88.2%, of which increment is equivalent to that of positive control (29.9%, interferon γ-treated cells). Human T-cells incubated with LX-2 cells showed significantly less cell viability in the presence of mesalamine than cells without mesalamine treatment (P

Background and aim: The three-panel marker, glypican-3, HSP70 and glutamine synthetase, is helpful for making a diagnosis of early hepatocellular carcinoma. To further characterize the diagnostic values of these three markers, we performed immunostaining studies in non-neoplastic hepatocytes (chronic hepatitis B) and hepatocellular carcinoma. Methods: Immunostaining was performed for all three markers. The cases were 60 needle biopsies of chronic hepatitis B (30 with low-grade activity and 30 with high-grade activity) and 40 hepatocellular carcinomas (HCCs). Results: Glypican-3 staining was detected in only one of the 60 chronic hepatitis B cases, and this case expressed moderate immunoreactivity for glypican-3 and showed high-grade inflammatory activity. HSP70 expression was not observed in any cases. Glutamine synthetase was strongly immunoreactive at peri-venular hepatocytes with cytoplasmic staining in all cases. These findings reflected a normal expression pattern. On the other hand, all three markers were positive in 15 (37.5%) HCCs. At least two of three markers were positive in 26 (65.0%). Moreover, all three markers were negative in 2 (5.0%) HCCs. Conclusions: The combination of these three markers is highly specific for detecting hepatocellular carcinoma. However, caution should be exercised because non-neoplastic hepatocytes in chronic hepatitis B with high necro-inflammatory activity tend to show focal positivity for glypican-3. Also we emphasize that all three markers can show negative immunoreactivity in HCC.

Although it is known that steatotic livers have a reduced ability to regenerate, most individuals with steatosis show generally benign prognosis. We hypothesized that a proliferative blockade in steatotic hepatocytes results in the compensatory expansion of hepatic progenitor cells (HPC) during fatty liver regeneration. Fifty-four cases of living donor liver transplantation (LDLT) with a liver biopsy performed at the postoperative 10th day were examined. HPC were counted by immunofluorescence histochemical dual-staining technique using cytokeratin 7 and Ki-67, and the replicative arrest of hepatocytes was assessed by p21 immunohistochemistry. The degree of ductular proliferation during regeneration 10 days after LDLT correlated both with the degree of steatosis and the number of HPC (P < 0.001). There was no difference in the average number of HPC and the replicative arrest index between donors with or without steatosis before LDLT (P = 0.111 and P = 0.062). However, degree of steatosis correlated with both the expansion of HPC and the replicative arrest index during liver regeneration 10 days after LDLT (P < 0.001 and P < 0.001, respectively). Moreover, increased replicative arrest was strongly associated with HPC expansion (P < 0.001). In conclusion, the compensatory expansion of HPC as a result of impaired hepatocyte replication occurred during steatotic liver regeneration after LDLT.

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) are the major adult liver cancers. The existence of combined hepatocellular-cholangiocarcinoma (CHC), a histopathologic intermediate form between HCC and CC, suggests phenotypic overlap between these tumors. Here, we applied an integrative oncogenomic approach to address the clinical and functional implications of the overlapping phenotype between these tumors. By performing gene expression profiling of human HCC, CHC, and CC, we identified a novel HCC subtype, i.e., cholangiocarcinoma-like HCC (CLHCC), which expressed cholangiocarcinoma-like traits (CC signature). Similar to CC and CHC, CLHCC showed an aggressive phenotype with shorter recurrence-free and overall survival. In addition, we found that CLHCC coexpressed embryonic stem cell–like expression traits (ES signature) suggesting its derivation from bipotent hepatic progenitor cells. By comparing the expression of CC signature with previous ES-like, hepatoblast-like, or proliferation-related traits, we observed that the prognostic value of the CC signatures was independent of the expression of those signatures. In conclusion, we suggest that the acquisition of cholangiocarcinoma-like expression traits plays a critical role in the heterogeneous progression of HCC. Cancer Res; 70(8); 3034–41. ©2010 AACR.

Human lung adenocarcinoma, the most prevalent form of lung cancer, is characterized by many molecular abnormalities. K-ras mutations are associated with the initiation of lung adenocarcinomas, but K-ras-independent mechanisms may also initiate lung tumors. Here, we find that the runt-related transcription factor Runx3 is essential for normal murine lung development and is a tumor suppressor that prevents lung adenocarcinoma. Runx3-/- mice, which die soon after birth, exhibit alveolar hyperplasia. Importantly, Runx3-/- bronchioli exhibit impaired differentiation, as evidenced by the accumulation of epithelial cells containing specific markers for both alveolar (that is SP-B) and bronchiolar (that is CC10) lineages. Runx3-/- epithelial cells also express Bmi1, which supports self-renewal of stem cells. Lung adenomas spontaneously develop in aging Runx3+/- mice ( approximately 18 months after birth) and invariably exhibit reduced levels of Runx3. As K-ras mutations are very rare in these adenomas, Runx3+/- mice provide an animal model for lung tumorigenesis that recapitulates the preneoplastic stage of human lung adenocarcinoma development, which is independent of K-Ras mutation. We conclude that Runx3 is essential for lung epithelial cell differentiation, and that downregulation of Runx3 is causally linked to the preneoplastic stage of lung adenocarcinoma.

Purpose Multidrug resistance-associated protein (MRP) 2 is a glutathione conjugate in the canalicular membrane of hepatocytes. Early graft damage after liver transplantation (LT) can result in alteration of MRP2 expression. The purpose of this study was to evaluate the relationship between the pattern of MRP2 alteration and graft outcome. Methods Forty-one paraffin-embedded liver graft tissues obtained by protocol biopsy within 2 months after LT; these were stained using monoclonal antibodies of MRP2. We selected 15 live donor biopsy samples as a control, that showed homogenous canalicular staining for MRP2. The pattern of canalicular MRP2 staining of graft was classified into 3 types: homogenous (type C0), focal (type C1), and no (type C2,) staining of the canaliculi. Results In total, 17.1% graft tissues were type C0, 36.6% were type C1, and 46.3% were type C2. The median operation time was longer in patients with type C2 (562.6 minutes) than in patients with type C0 (393.8 minutes) (P = 0.038). The rates of posttransplant complications were higher in patients with type C2 (100%) than in patients with type C0 (42.9%) and C1 (73.3%) (P < 0.001). Conclusion MRP2 expression pattern was altered in 82.9% after LT. The pattern of MRP2 alteration was associated with longer operation time and higher rates of post-LT complications.

Background/Aims: Hepatoblastoma (HB) and hepatocellular carcinoma (HCC) are the two most common primary malignant liver tumours in children. Hepatic progenitor cells have been described and can be stained with K19, EpCAM and CD117. We investigated the morphology and staining patterns of primary liver tumours in Asian children. Methods: Four pathologists studied slides from 39 paediatric patients from Vietnam and Korea aged 8 months to 16 years. We performed immunohistochemical stains for K19, EpCAM and CD117, and polymerase chain reaction for tissue hepatitis B virus (HBV) DNA and hepatitis C virus (HCV) RNA. Results: There was agreement on the diagnosis of 24 cases of HCC and 10 cases of HB (one recurrent case). The diagnosis was split for six cases (HCC/HB). All 20 cases of HCC tested were HBV DNA+ and HCV RNA−. All nine cases of HB tested were HBV DNA−, while one was HCV RNA+. Of four HCC/HB cases tested, three were HBV DNA+ and all were HCV RNA−. By immunohistochemistry, 8/24 (33%) cases of HCC were K19+ and 18/24 (75%) were EpCAM+, 5/10 (50%) cases of HB were K19+ and 7/10 (70%) were EpCAM+ and 3/6 (50%) cases of HCC/HB were K19+ and 5/6 (83%) were EpCAM+. CD117 was negative in all 38 cases tested. Paediatric HCC has a morphology different from adult HCC, sometimes resembling HB, and a larger proportion of paediatric tumours have progenitor cell features. Conclusions: HB and HCC in children may represent malignant transformation at an early stage in the cellular lineage and often arise from hepatic progenitor cells.

Thioredoxin (Trx) is a multifunctional redox protein that has growth-promoting and anti-apoptotic effects on cells and protects cells from endogenous and exogenous free radicals. Recently, altered expression of Trx has been reported in various cancers. In the present study, we investigated altered expression of Trx at the precancerous and carcinogenic phases during cholangiocarcinogenesis in a hamster cholangiocarcinoma (ChC) model, using semiquantitative immunohistochemical and Western blot analyses. Moreover, to determine if the results correlated well with those in human ChCs, we carried out a comparative immunohistochemical study for Trx in tissue-arrayed human ChCs with different grades of tumor cell differentiation. Trx was found highly expressed in the cytoplasm of dysplastic bile ducts with highly abnormal growth patterns and ChCs irrespective of tumor type or tumor cell differentiation. Overexpression of Trx at the precancerous and carcinogenic phases was further supported by significant elevation of Trx protein in Western blotting. The results from the hamster ChCs were in good agreement with those from human ChCs. Our results strongly suggested that the redox regulatory function of Trx plays an important role in bile duct cell transformation and tumor progression during cholangiocarcinogenesis.

Background and aims: The expression pattern of high affinity neurotrophin receptors dichotomizes the clinicopathologic characteristics of neuroblastoma (NB). The effects mediated by trkA have been documented in many experimental systems, whereas the effects mediated by trkB have only been investigated in limited studies. Methods: We established a new cell line of NB, “SNU-NB1”, by primary culture and the effects mediated by trkB were addressed in this cell line. Results: NB cells in primary culture showed amplification of N-myc and expression of trkB. Activation of trkB in primary culture by brain-derived neurotrophic factor (BDNF) was followed by activation of extracellular signal-regulated kinase1/2 (ERK1/2), growth inhibition, neuronal differentiation, and N-myc down-regulation. However, fully transformed SNU-NB1 cells lost BDNF sensitivity while being tumorigenic in vivo. Restoration of trkB expression in SNU-NB1 cells, by retroviral infection of trkB, induced differentiation and inhibition of growth in the cells following BDNF treatment. Although the prognosis of neuroblastomas is different according to the status of trkA and trkB mRNA expressions, the cytologic effects mediated by trkB per se are similar to those by trkA in NB. Conclusion: The patterns of trkA and trkB mRNA expression were markers for different lineages of transformed cells but were inconsistent with the clinical behavior.

Although many studies on the risk factors and their carcinogenesis in adult hepatocellular carcinoma (HCC) have been reported, they remain poorly understood in childhood HCC. A retrospective study of 42 HCC cases in Asian children was conducted. Hepatitis B virus (HBV)-DNA in HCC tissues was detected in 36 of 42 (86%) cases tested, while no hepatitis C virus (HCV)-RNA was detectable in any of HCCs. Twenty of 36 (56%) HCC cases were accompanied by cirrhosis. Surprisingly, very high prevalence of the HBV pre-S deletion mutant was recognized in 27 of 30 (90%) HCCs examined. They occurred most frequently in pre-S2 (20/27, 74%) followed by pre-S1 (5/27, 18.5%), and both pre-S1/S2 (2/27, 7.4%). Interestingly, the pre-S2 mutant consistently appeared with deletion at nt 4-57 in all of the 20 cases with the pre-S2 mutant (100%) and within this locus in the two cases with both pre-S-1/S2 mutants. Type II ground-glass hepatocytes in non-tumorous livers were seen in 15 of the 22 HCCs with the pre-S2 deletion mutant (68%). This hotspot mutation in the pre-S2 was further confirmed by complete genomic sequence of HBV in a Japanese boy who eventually developed HCC. Our result strongly suggests that HBV is a major contributor to the development of HCC in Asian children. The HBV pre-S2 deletion mutant at nt 4-57 which has a CD8 T-cell epitope could be responsible for the emergence and aggressive outcome of childhood HCC. Determination of this hotspot mutation in the pre-S2 region could be a useful index for predicting the clinical outcome of HCC development.

Hepatocellular carcinoma (HCC) is the fifth most frequent cause of cancer deaths in males and was the third most frequent cause of cancer deaths in 2007 throughout the world. The incidence rate is 2-3 times higher in developing countries than in developed countries. Animal models have enabled study of the mechanism of HCC and the development of possible strategies for treatment. Diethylnitrosamine (DEN) is a representative chemical carcinogen with the potential to cause tumors in various organs, including the liver, skin, gastrointestinal tract, and respiratory system. Specifically in HCC, DEN is a complete carcinogen. Many lines of evidence have demonstrated a relationship between carcinogenesis and cell cycle regulation. In this study we found that cell cycle regulatory proteins were critically involved in cancer initiation and promotion by DEN. Cyclin D1, cyclin E, cdk4, and p21(CIP1/WAF1) are factors whose expression levels may be useful as criteria for the classification of hepatic disease. In particular, cdk4 had a pivotal role in the transition to the neoplastic stage. In conclusion, we suggest that changes in the level of cdk4 may be useful as a biomarker for detection of HCC.

Biliary intraepithelial neoplasia (BilIN) is the premalignant lesion of extrahepatic cholangiocarcinoma (EHC), and there are no published data regarding epigenetic changes throughout disease progression from normal biliary epithelia to BilIN to EHC. The objective of this study was to identify the occurrence of CpG island hypermethylation and repetitive DNA hypomethylation in BilIN. A total of 50 EHCs, 31 BilINs, and 31 normal cystic duct samples were analyzed for their methylation status in seven genes and two repetitive DNA elements. The number of methylated genes increased with disease progression (normal bile duct, 0.6; BilIN, 2.0; EHC, 3.6; P < 0.001). The methylation level of examined genes was significantly higher in BilIN than in normal samples (TMEFF2, HOXA1, NEUROG1, and RUNX3, P < 0.05) and in EHC than in BilIN samples (TMEFF2, HOXA1, NEUROG1, RUNX3, RASSF1A, and APC, P < 0.05). Long interspersed nucleotide element-1 (LINE-1) and juxtacentromeric satellite 2 (SAT2) methylation levels were markedly lower in EHC than in normal duct and BilIN samples, and BilIN samples showed a decrease of SAT2 methylation levels but no decrease of LINE-1 methylation levels compared to normal samples. These findings suggest that most of cancer-specific CpG island hypermethylation occur in the stage of BilIN and that CpG island hypermethylation seems to occur earlier than repetitive DNA element hypomethylation.

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.

Background and aim: Large scale studies on protein targeting for hepatocellular carcinoma (HCC) are limited and the clinical implications of targeted proteins have not been fully investigated. We studied the expression of three candidate antigens, glypican-3(GPC3), human melanoma antigen-1(MAGE-1) and alpha-fetoprotein (AFP) in HCC on large number samples. Methods: We performed immunohistochemistry using tissue microarray on 420 HCC samples and 120 non-tumorous hepatic tissues. Results: GPC3 staining was observed in 250 (62.3%) of 401 HCC cases, while none of the control group had positive staining. The expression of MAGE-1 was 9.75%, 7.1% and 18.8% with the three different antibodies. None of the control group was stained with MAGE-1, with two antibodies. Control cases were more frequently stained than HCC with two different AFP antibodies. Microscopic vascular invasion, and T-stage correlated with the expression of GPC3. Vascular and other organ invasion correlated with MAGE-1 expression. GPC-3+ HCC was associated with a short survival rate. Conclusions: GPC3 and MAGE-1 are useful biomarkers indicating tumor aggressiveness as well as potent target proteins for HCC immunotherapy. AFP staining had a high false positivity and further evaluation is required.

The purpose of this study was to retrospectively evaluate the diagnostic performance of dynamic gadobenate dimeglumine-enhanced MRI with explant pathologic correlation in the detection of hepatocellular carcinoma (HCC) in patients undergoing liver transplantation.Forty-seven patients (28 men, 19 women; mean age, 49 years) underwent dynamic gadobenate dimeglumine-enhanced MRI within 3 months before primary liver transplantation. Dynamic imaging was performed before (unenhanced) and after (hepatic arterial, portal venous, equilibrium, and 1-hour delayed phases) IV bolus administration of gadobenate dimeglumine at 0.1 mmol/kg body weight. Retrospective image analysis to detect HCC nodules was performed independently by two abdominal radiologists who had no pathologic information. On a per-nodule basis, the sensitivity and positive predictive value were calculated for the two observers. Sensitivity and specificity in the diagnosis of HCC also were evaluated. Fisher's exact test was performed to determine whether there was a detection difference between HCC nodules 1 cm in diameter or larger and nodules smaller than 1 cm and to evaluate the differences in causes of false-positive MRI findings based on lesion size (or= 1 cm vs1 cm).Twenty-seven patients had 41 HCCs. In HCC detection, gadobenate dimeglumine-enhanced MRI had a sensitivity of 85% (35 of 41 HCCs) and a positive predictive value of 66% (35 of 53 readings) for observer 1 and a sensitivity of 80% (33 of 41 HCCs) and a positive predictive value of 65% (34 of 52 readings) for observer 2. For both observers, sensitivity in the detection of HCCs 1 cm in diameter and larger (91-94%) was significantly different (p0.05) from that in detection of HCCs smaller than 1 cm (29-43%). Nonneoplastic arterial hypervascular lesions more often caused false-positive diagnoses of lesions smaller than 1 cm in diameter (80-86%) on MR images than of those 1 cm in diameter and larger (0-25%). The difference was statistically significant (p0.05) for both observers. In diagnosis, gadobenate dimeglumine-enhanced MRI had a sensitivity of 87% (20 of 23 patients) and a specificity of 79% (19 of 24 patients) for both observers.Dynamic gadobenate dimeglumine-enhanced MRI has a sensitivity of 80-85% and a positive predictive value of 65-66% in the detection of HCC. The technique, however, is of limited value for detecting and characterizing lesions smaller than 1 cm in diameter.

Context.—CpG island hypermethylation is attracting attention because of its importance as a tumor marker and its potential mechanism for the development of human cancers. Extrahepatic cholangiocarcinoma has been poorly investigated with respect to CpG island hypermethylation, and the number of genes known to be methylated in extrahepatic cholangiocarcinomas is fewer than 20.Objective.—To generate methylation profiles of 24 CpG island loci in extrahepatic cholangiocarcinomas, to correlate methylation findings with clinicopathologic findings, and to compare these findings with those of intrahepatic cholangiocarcinomas.Design.—Sixty-three extrahepatic cholangiocarcinomas and 48 intrahepatic cholangiocarcinomas were investigated for hypermethylation in 24 CpG island loci by using methylation-specific polymerase chain reaction.Results.—A total of 61 (96.8%) of 63 extrahepatic cholangiocarcinomas showed hypermethylation in at least one of the examined loci, and a high methylation frequency was seen in HOXA1 (95.2%), HPP1 (69.8%), and NEUROG1 (61.9%). The number of methylated CpG island loci was greater in extrahepatic cholangiocarcinomas with nodal metastasis than in those without nodal metastasis (P = .047), and hypermethylation of TIG1 was closely associated with nodal metastasis of extrahepatic cholangiocarcinomas (P = .007). CDH1 and NEUROG1 were more frequently methylated in extrahepatic cholangiocarcinoma than in intrahepatic cholangiocarcinoma, whereas CHFR, GSTP1, IGF2, MGMT, MINT31, p14, and RBP1 were more frequently methylated in intrahepatic cholangiocarcinoma: the differences was statistically significant (P < .05).Conclusions.—A close relationship exists between CpG island hypermethylation and nodal metastasis of extrahepatic cholangiocarcinomas. Methylation profiles of extrahepatic cholangiocarcinomas are somewhat similar to but distinct from those of intrahepatic cholangiocarcinomas.

Wnt signaling was implicated in pulmonary and renal fibrosis. Since Wnt activity is enhanced in liver cirrhosis, Wnt signaling may also participate in hepatic fibrogenesis. Thus, we determined if Wnt signaling modulates hepatic stellate cell (HSC) activation and survival. Wnt3A treatment significantly activated human HSCs, while this was inhibited in secreted frizzled-related protein 1 (sFRP1) overexpressing cells. Wnt3A treatment significantly suppressed TRAIL-induced apoptosis in control HSCs versus sFRP1 over-expressing cells. Particularly, caspase 3 was more activated in sFRP1 over-expressing cells following TRAIL and Wnt3A treatment. These observations imply that Wnt signaling promotes hepatic fibrosis by enhancing HSC activation and survival.

In cholestasis, bile acids induce hepatocyte apoptosis, while activation of hepatic stellate cells (HSCs) results in fibrosis. Since transforming growth factor-beta (TGF-beta) is a critical mediator in this process, we hypothesized that bile acids may participate in TGF-beta-mediated HSC activation in cholestasis. Bile acid treatment increased TGF-beta transcription in hepatocytes, while the total TGF-beta concentration in culture media rapidly decreased following bile acid treatment. Bile acid treatment promptly induced thrombospondin-1 expression in hepatocytes, which is a potent activator of latent TGF-beta, whereas this induction was not observed in bile acid-treated HSCs. HSCs co-cultured with hepatocytes showed a significantly higher level of Smad2 phosphorylation and collagen alpha1 synthesis following bile acid treatment than cells cultured without hepatocytes. Moreover, this enhanced collagen synthesis was significantly inhibited in the presence of TGF-beta receptor inhibitor. These observations imply that bile acids induce thrombospondin-1 expression in hepatocytes, which activates latent TGF-beta leading to HSC activation.

Summary There is no agreement regarding the treatment of early allograft rejection (EAR) in adult living donor liver transplantation (LDLT). A protocol biopsy was performed in 62 adult LDLT recipients. Twenty-one patients (33.9%) had histological evidence of EAR. Of these, 14 patients had biochemical abnormalities and seven patients had no associated biochemical abnormalities. None of the seven patients with subclinical EAR (11.3% of the entire study population) were treated, and no subsequent rejection was observed. Gender mismatch (female-to-male) was the single independent risk factor for histological EAR [odds ratio (OR) = 13.458; 95% confidence interval (CI), 1.836–98.649] and the cumulative probability for a subsequent rejection was higher in patients with EAR (OR = 11.085; 95% CI, 1.221–100.654). However, the actuarial 1 year patient and graft survival rate in patients with EAR (81.0% and 85.5%) were similar to those without EAR (92.7% and 97.25%; P = 0.127 and 0.302, respectively). The presence of an initial biochemical abnormality was an independent risk factor for both a decreased patient survival (OR = 5.827; 95% CI, 1.095–31.017; P = 0.039) and graft loss (OR = 20.646; 95% CI, 2.044–208.524; P = 0.010). Subsequent rejection developed more frequently in patients with EAR. However, the survival is not determined by the presence of EAR but by the presence of a biochemical abnormality.

Purpose: Lung cancer develops through a multistage process involving the accumulation of diverse genetic alterations. To gain an understanding of the roles played by tumor suppressor gene proteins and proliferating cell nuclear antigen (PCNA) in chemical carcinogen-induced mouse lung tumorigenesis, we examined the expression of retinoblastoma protein (Rb), p53, and PCNA in normal lung tissues and urethane-induced mouse lung tumors. Materials and Methods: ICR mice were given urethane by intra-peritoneal injection, and sacrificed at 5, 13, 21, 31, and 37 weeks following treatment. Sequential morphological changes and the immunohistochemical expression of Rb protein, p53, and (PCNA), during mouse lung tumorigenesis, were examined. Results: During the carcinogenesis, sequential histological changes from hyperplasia of type II pneumocytes, to adenomas, and ultimately to overt adenocarcinomas were noted. Intense nuclear staining of the Rb protein was observed in normal and hyperplastic alveolar epithelial cells and adenomas. In adenocarcinomas, the Rb protein expression was significantly diminished. The p53 mutant protein was not detected in any lesion. The PCNA labeling index increased along with the advance in the histological grade. Conclusion: The above results indicate that mouse pulmonary adenocarcinomas develop through premalignant lesions, and down-regulation of the Rb protein expression may be implicated in the urethane-induced mouse lung tumorigenesis. In addition, the PCNA labeling index may reflect the malignant potential during the tumor progression.

Background More knowledge about genetic and molecular features of cholangiocarcinoma is needed to develop effective therapeutic strategies. We investigated the clinical and pathological significance of ROS1 expression in intrahepatic cholangiocarcinoma. Methods One hundred ninety-four patients with curatively resected intrahepatic cholangiocarcinoma were included in this study. Tumor tissue specimens were collected and analyzed for ROS1 gene rearrangement using fluorescence in situ hybridization (FISH) and ROS1 protein expression using immunohistochemistry (IHC). Results ROS1 immunohistochemistry was positive (moderate or strong staining) in 72 tumors (37.1 %). ROS1 protein expression was significantly correlated with well differentiated tumors, papillary or mucinous histology, oncocytic/hepatoid or intestinal type tumors, and periductal infiltrating or intraductal growing tumors (vs. mass-forming cholangiocarcinoma). ROS-expressing tumors were associated with better disease-free survival (30.1 months for ROS1 expression (+) tumors vs. 9.0 months for ROS1 (−) tumors, p = 0.006). Moreover, ROS1 expression was an independent predictor of better disease-free survival in a multivariate analysis (HR 0.607, 95 % CI 0.377–0.976; p = 0.039). Although break-apart FISH was successfully performed in 102 samples, a split pattern indicative of ROS1 gene rearrangement was not found in the examined samples. Conclusion ROS1 protein expression was associated with well-differentiated histology and better survival in our patients with resected intrahepatic cholangiocarcinoma. ROS1 gene rearrangement by break-apart FISH was not found in the examined samples.

BackgroundThere is no consensus on the best method of preventing postoperative pancreatic fistula (POPF) after pancreaticoduodenectomy (PD). This multicentre, parallel group, randomized equivalence trial investigated the effect of two ways of pancreatic stenting after PD on the rate of POPF.MethodsPatients undergoing elective PD or pylorus-preserving PD with duct-to-mucosa pancreaticojejunostomy were enrolled from four tertiary referral hospitals. Randomization was stratified according to surgeon with a 1 : 1 allocation ratio to avoid any related technical factors. The primary endpoint was clinically relevant POPF rate. Secondary endpoints were nutritional index, remnant pancreatic volume, long-term complications and quality of life 2 years after PD.ResultsA total of 328 patients were randomized to the external (164 patients) or internal (164) stent group between August 2010 and January 2014. The rates of clinically relevant POPF were 24·4 per cent in the external and 18·9 per cent in the internal stent group (risk difference 5·5 per cent). As the 90 per cent confidence interval (−2·0 to 13·0 per cent) did not fall within the predefined equivalence limits (−10 to 10 per cent), the clinically relevant POPF rates in the two groups were not equivalent. Similar results were observed for patients with soft pancreatic texture and high fistula risk score. Other postoperative outcomes were comparable between the two groups. Five stent-related complications occurred in the external stent group. Multivariable analysis revealed that soft pancreatic texture, non-pancreatic disease and high body mass index (23·3 kg/m2 or above) predicted clinically relevant POPF.ConclusionExternal stenting after PD was associated with a higher rate of clinically relevant POPF than internal stenting. Registration number: NCT01023594 (https://www.clinicaltrials.gov).

The rhizomes of Cimicifuga species, including Cimicifuga heracleifolia (CH), have been widely used as antipyretic, analgesic, and anti-inflammatory agents in oriental countries. However, information regarding its toxicity, especially long-term toxicity and genotoxicity, is limited. Therefore, we performed the subchronic toxicity and genotoxicity assays of the CH extract in accordance with the test guidelines published by the Organization for Economic Cooperation and Development. In a 13-week repeat-dose oral toxicity study, the CH extract did not influence body weight, food/water consumption, mortality, clinical signs, and urinalysis throughout the study. Noteworthy, the CH extract groups exhibited increased liver weights along with serum alanine transaminase activity rise at doses of 667 and 2000 mg/kg in females. No-observed-adverse-effect-level of the CH extract administered orally was concluded to be 2000 mg/kg body weight/day for male rats and 222 mg/kg body weight/day for female rats. The CH extract did not exert a mutagenic or clastogenic effect in Ames test, in vitro chromosome aberration assay and in vivo micronucleus assay. Overall findings of the subchronic toxicity study indicate for the first time that the CH extract may possess hepatotoxic potential in female rats, suggesting that further mechanistic studies should be performed to have more conclusive results on hepatotoxic potential of the CH extract.