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Intrahepatic cholangiocarcinoma (ICC) is a rare but fatal tumor. The isocitrate dehydrogenase 1 and 2 (IDH1/2) genes are known to be mutated in ICC. IDH1/2 mutations tend to be accompanied by enhanced hypermethylation at a subset of genomic loci. We sought to clarify the clinicopathological features, including prognostic value, of ICCs with IDH1/2 mutation-associated hypermethylation at a subset of genes. The mutation status of IDH1/2 and methylation status of 30 gene CpG island loci were analyzed in 172 cases of ICC using pyrosequencing and the MethyLight assay, respectively. The mutation status of IDH1/2 was correlated with clinicopathological features and the DNA methylation status at 30 gene loci. Then, the clinicopathological characteristics were analyzed regarding three-tiered methylation statuses in genes showing IDH1/2 mutation-associated methylation. IDH1/2 mutations were found in 9.3% of ICCs, and IDH1/2-mutated tumors were associated with the histological subtype, including the bile ductular type and small duct type, and poor differentiation. Eight DNA methylation markers showed associations with IDH1/2 mutations, and ICCs with > 5/8 methylated markers were associated with the bile ductular type or small duct type, absence of mucin production, absence of biliary intraepithelial neoplasia, and presence of chronic liver disease. > 5/8 methylated markers were an independent prognostic marker associated with better survival in both cancer-specific survival and recurrence-free survival. In summary, by analyzing the association between IDH1/2 mutations and DNA methylation in individual genes, we developed a panel of DNA methylation markers that were significantly associated with IDH1/2 mutations and were able to identify a subset of ICC with better clinical outcomes.

Intrahepatic cholangiocarcinoma is a complex disease with three different histologic subtypes, the large duct, small duct, and bile ductular types. In the present study, we elucidated whether the three histological subtypes have differences in their methylation profiles and developed a DNA methylation marker that might help identify a subset of ICC with a different prognosis. We screened 113 promoter CpG island loci against 10 cases of intrahepatic cholangiocarcinoma and normal cystic ducts using the MethyLight assay and selected 30 CpG island loci with cancer-associated hypermethylation. Then, we analyzed 172 intrahepatic cholangiocarcinomas for the methylation state at these 30 loci. Six loci, including DLEC1, were more frequently methylated in the bile ductular type and small duct type, whereas six loci were more frequently methylated in the large duct type. Of these 30 loci, DLEC1 methylation was found mainly in the bile ductular type and small duct type but rarely in the large duct type. DLEC1 methylation was significantly associated with a better clinical outcome in intrahepatic cholangiocarcinomas of the small duct type but not of the bile ductular type. DLEC1 methylation was an independent prognostic variable in both cancer-specific survival and recurrence-free survival. For patients with intrahepatic cholangiocarcinoma of the small duct type (n = 68), DLEC1 methylation was found in 26 (38.2%) and was associated with a better clinical outcome for both cancer-specific survival and recurrence-free survival. Our findings suggest that DLEC1 methylation can be utilized to identify a subset with a better prognosis in intrahepatic cholangiocarcinomas of the small duct type.

Background: Ampulla of Vater cancer (AoV Ca) is a rare tumor, and its adjuvant treatment has not been established. The purpose of this study was to find out prognostic factors including host immunity and role of adjuvant treatment in AoV Ca., Methods and Findings: We reviewed 227 AoV Ca patients with curative resection. Clinical characteristics, adjuvant treatment, disease-free survival (DFS) and overall survival (OS) were analyzed. Among all patients, 63.9, 36.1 and 33.9% had T1/T2, T3/T4 stage and lymph node-positive disease (LN+), respectively. OS of all patients was 90.9 months (95% CI: 52.9-129.0). OS was different according to neutrophil-to-lymphocyte ratio (HR 1.651, 95% CI: 1.11-2.47), platelet-to-lymphocyte ratio (HR 1.488, 95% CI: 1.00-2.21) and systemic inflammatory index (HR 1.669, 95% CI: 1.13-2.47). In multivariate analysis, adverse prognostic factors for OS included vascular invasion (HR 2.571, 95% CI: 1.20-5.53) and elevated CA 19-9 (HR 1.794, 95% CI: 1.07-3.05). A total of 104 patients (46.3%) received adjuvant treatment (25 out of 111of T1/T2 & LN (-), 79 out of 116 of T3/T4 or LN (+)). In T3/T4 or LN (+) stage, adjuvant CCRT with maintenance chemotherapy provided the longest OS (5-year OS rate: 47.0 vs. 41.4%)., Conclusions: Vascular invasion and elevated CA 19-9 were adverse prognostic factors in resected AoV Ca. In T3/T4 or LN (+) stage, adjuvant CCRT with maintenance chemotherapy provided the best survival outcome. Adjuvant treatment should be further defined in AoV Ca, especially with poor prognostic factors.

Intraductal papillary mucinous neoplasm (IPMN) is a common precursor of pancreatic cancer (PC). Much clinical attention has been directed toward IPMNs due to the increase in the prevalence of PC. The diagnosis of IPMN depends primarily on a radiological examination, but the diagnostic accuracy of this tool is not satisfactory, necessitating the development of accurate diagnostic biomarkers for IPMN to prevent PC. Recently, high-throughput targeted proteomic quantification methods have accelerated the discovery of biomarkers, rendering them powerful platforms for the evolution of IPMN diagnostic biomarkers. In this study, a robust multiple reaction monitoring (MRM) pipeline was applied to discovery and verify IPMN biomarker candidates in a large cohort of plasma samples. Through highly reproducible MRM assays and a stringent statistical analysis, 11 proteins were selected as IPMN marker candidates with high confidence in 184 plasma samples, comprising a training (n = 84) and test set (n = 100). To improve the discriminatory power, we constructed a six-protein panel by combining marker candidates. The multimarker panel had high discriminatory power in distinguishing between IPMN and controls, including other benign diseases. Consequently, the diagnostic accuracy of IPMN can be improved dramatically with this novel plasma-based panel in combination with a radiological examination.

Aims: To address whether napsin A is useful for identifying metastatic adenocarcinomas of pulmonary origin., Methods and Results: Fifty-four cases of adenocarcinoma that metastasized from the lungs to various sites and 1762 cases of carcinoma from various organs were immunostained for napsin A, TTF-1, CK7, CK20 and CDX2 using tissue microarray. The expression patterns of napsin A and TTF-1 in metastatic pulmonary adenocarcinomas were compared with matched primary lung tumours. Napsin A and TTF-1 were expressed in 87.0% and 81.5% of the metastatic pulmonary adenocarcinomas, respectively. Although there was no significant difference in the positivity of napsin A and TTF-1 as a single marker in metastatic pulmonary adenocarcinomas, the expression scores for napsin A were much higher than those for TTF-1 (P < 0.001). Moreover, the positivity and expression scores of napsin A in primary pulmonary adenocarcinomas were maintained in metastatic adenocarcinomas better than TTF-1. Most non-pulmonary adenocarcinomas were negative for napsin A, except for renal cell carcinomas (13.4%), ovarian adenocarcinomas (7.1%) and uterine endometrial adenocarcinomas (14.5%). In particular, clear cell adenocarcinomas of ovary (68.8%) and uterus (66.7%) frequently expressed napsin A., Conclusions: These data suggest that napsin A may be a useful marker for identifying metastatic adenocarcinomas of pulmonary origin in combination with TTF-1., (© 2014 John Wiley & Sons Ltd.)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background & Aims: Chronic oxidative stress from reactive oxygen species (ROS) produced by the mitochondria promotes hepatocarcinogenesis and tumor progression. However, the exact mechanism by which mitochondrial ROS contributes to tumor cell invasion is not known. We investigated the role of ROS modulator 1 (Romo1) in hepatocellular carcinoma (HCC) development and tumor cell invasiveness., Methods: We performed real-time, semi-quantitative, reverse transcriptase polymerase chain reaction; invasion and luciferase assays; and immunofluorescence and immunohistochemical analyses. The formation of pulmonary metastatic nodules after tumor cell injection was tested in severe combined immunodeficient mice. We analyzed Romo1 expression in HCC cell lines and tissues (n = 95)., Results: Expression of Romo1 was increased in HCC cells, compared with normal human lung fibroblast cells. Exogenous expression of Romo1 in HCC cells increased their invasive activity, compared with control cells. Knockdown of Romo1 in Hep3B and Huh-7 HCC cells reduced their invasive activity in response to stimulation with 12-O-tetradecanoylphorbol-13-acetate. Levels of Romo1 were increased compared with normal liver tissues in 63 of 95 HCC samples from patients. In HCC samples from patients, there was an inverse correlation between Romo1 overexpression and patient survival times. Increased levels of Romo1 also correlated with vascular invasion by the tumors, reduced differentiation, and larger tumor size., Conclusions: Romo1 is a biomarker of HCC progression that might be used in diagnosis. Reagents that inhibit activity of Romo1 and suppress mitochondrial ROS production, rather than eliminate up-regulated intracellular ROS, might be developed as cancer therapies., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Objective: Although hepatocellular carcinoma (HCC) demonstrates characteristic hypervascularity, some HCCs have a hypovascular pattern on computed tomography (CT). Cytokeratin 19 (CK19) is a marker for the biliary phenotype reflecting a poor prognosis. We assessed the prognostic implications of tumor vascularity and its association with CK19 expression in HCC., Methods: Patients that underwent surgical resection for HCC were included. Tumor vascularity was evaluated according to the arterial enhancement patterns on CT scans and CK19 expression was evaluated by using tissue microarray methods., Results: One hundred and forty patients were included. Their median follow-up duration was 55.0 months, and 92 (65.7%) patients had tumor recurrence. Forty-five patients (30.6%) had hypovascular HCC at the time of diagnosis, and they showed a significantly higher CK19 expression rate (32.5% vs. 8.2%, P = 0.001) and earlier recurrence rate within 6 months (hazard ratio (HR), 2.301; P = 0.025) compared to the patients with hypervascular HCCs. Hypovascularity (HR, 1.694; P = 0.045) was an independent risk factor for short overall survival., Conclusions: Hypovascular HCCs were associated with early recurrence and short overall survival, and CK19 was more frequently expressed in hypovascular HCC than in hypervascular tumors. Therefore, tumor vascularity on CT images might be utilized in determining the prognosis of patients with HCCs.

Aims: CD151 is known to be implicated in cancer progression and metastasis. The aim was to evaluate the expression of CD151 in clear cell renal cell carcinoma (CCRCC) and to assess its prognostic significance., Methods and Results: The expression of CD151 was evaluated in 489 cases of CCRCC by immunohistochemistry. Immunoreactivity was classified into four categories (minimal, 0-10% positive cells; focal, 10-50% positive cells; diffuse moderate, >50% positive cells with moderate staining intensity; diffuse strong, >50% positive cells with strong staining). To determine the statistical significance of CD151 expression in CCRCC, all cases were divided into two groups based on their CD151 expression level: a CD151-low group (n=257; minimal and focal) and a CD151-high group (n=232; diffuse). Expression of CD151 was correlated positively with pT, pN, pM categories, pathological tumour-node-metastasis (pTNM) stage, nuclear grade, tumour size and patient's age. The CD151-high group had significantly shorter cancer-specific survival (P<0.001) and progression-free survival (P<0.001) times. Furthermore, multivariate analysis showed that CD151 expression was an independent predictor for tumour progression in patients with CCRCC (P=0.040)., Conclusions: High CD151 expression is associated with advanced stage and high nuclear grade in CCRCC. CD151 is a prognostic marker for tumour progression in CCRCC patients., (© 2011 Blackwell Publishing Limited.)