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Accurate staging of liver fibrosis is crucial to guide therapeutic decisions for patients with nonalcoholic fatty liver disease (NAFLD). Digital image analysis has emerged as a promising tool for quantitative assessment of fibrosis in chronic liver diseases. We sought to determine the relationship of histologic fibrosis stage with fiber amounts quantified in liver biopsy specimens for the better understanding of NAFLD progression. We measured area ratios of collagen and elastin fibers in Elastica van Gieson-stained biopsy tissues from 289 patients with NAFLD from four hospitals using an automated computational method and examined their correlations with Brunt's fibrosis stage. As a secondary analysis, we performed multivariable logistic regression analysis to assess the associations of the combined area ratios of collagen and elastin with noninvasive fibrosis markers. The combined fiber area ratios correlated strongly with Brunt's stage (Spearman correlation coefficient, 0.78; P < 0.0001), but this relationship was nonlinear (P = 0.007) with striking differences between stage 4 (median area ratios, 12.3%) and stages 0-3 (2.1%, 2.8%, 4.3%, and 4.8%, respectively). Elastin accumulation was common in areas of thick bridging fibrosis and thickened venous walls but not in areas of perisinusoidal fibrosis. The highest tertile of the combined fiber area ratios was associated with the fibrosis-4 index and serum type IV collagen 7s domain (7s collagen) levels, whereas the upper two tertiles of the fiber amounts significantly associated with body mass index, aspartate aminotransferase, and 7s collagen in the multivariable analysis. Conclusion: Quantitative fibrosis assessment reveals a nonlinear relationship between fibrosis stage and fiber amount, with a marked difference between stage 4 and stage 3 and much smaller differences among stages 0-3, suggesting a heterogeneity in disease severity within NAFLD-related cirrhosis. (Hepatology Communications 2018;2:58-68).

Malignant melanoma is one of the lethal malignant tumors worldwide. Previously we reported that adenylate cyclase-associated protein 2 (CAP2), which is a well-conserved actin regulator, was overexpressed in hepatocellular carcinoma; however, CAP2 expression in other clinical cancers remains unclear. The aim of the current study was to clarify the clinicopathological significance of CAP2 overexpression in malignant melanoma. Immunohistochemical analyses revealed that many melanoma cells exhibited diffuse cytoplasmic expression of CAP2, whereas no normal melanocytes showed detectable immunostaining for CAP2. A high level of CAP2 expression was seen in 14 of 50 melanomas and was significantly correlated with greater tumor thickness and nodular melanoma subtypes. In addition, a high level of CAP2 expression was associated with poor overall survival in univariate and multivariate analyses. For 13 patients, samples of primary and metastatic melanoma tissue were available: four patients exhibited higher levels of CAP2 expression in metastatic tumor compared to the primary site, whereas no patient showed lower levels of CAP2 expression in metastatic melanomas. Our findings show that CAP2 overexpression is a novel prognostic marker in malignant melanoma and that CAP2 expression seems to increase stepwise during tumor progression, suggesting the involvement of CAP2 in the aggressive behavior of malignant melanoma.

In pancreatic ductal adenocarcinoma (PDAC), features of epithelial-mesenchymal transition (EMT) are often seen in tumor tissue, and such features correlate with poor prognosis. Solitary infiltration of tumor cells represents a morphological phenotype of EMT, and we previously reported that a high degree of solitary cell infiltration correlates with EMT-like features, including reduced E-cadherin and elevated vimentin levels. Using solitary cell infiltration to evaluate the degree of EMT, gene-expression profiling of 12 PDAC xenografts was performed, and SMAD3 was identified as an EMT-related gene. Immunohistochemistry using clinical specimens (n=113) showed that SMAD3 accumulated in the nuclei of tumor cells, but was not detected in most epithelial cells in the pancreatic duct. Moreover, SMAD3 upregulation correlated with malignant characteristics, such as higher tumor grade and lymph node metastasis, as well as with EMT-like features. SMAD4, which plays a key role in transforming growth factor-β (TGF-β) signaling, is inactivated in approximately half of PDAC cases. In this study, the nuclear accumulation of SMAD3 was immunohistochemically detected even in SMAD4-negative cases. SMAD3 knockdown resulted in upregulated E-cadherin, downregulated vimentin, and reduced cell motility in pancreatic cancer cells regardless of SMAD4 status. In addition, TGF-β-treatment resulted in EMT induction in cells carrying wild-type SMAD4, and EMT was suppressed by SMAD3 knockdown. Patients with upregulated SMAD3 and a high degree of solitary cell infiltration had shorter times to recurrence and shorter survival times after surgery, and multivariate analysis showed that both factors were independent prognostic factors linked to unfavorable outcomes. These findings suggest that SMAD3 in PDAC is involved in the promotion of malignant potential through EMT induction in tumor cells regardless of SMAD4 status and serves as a potential biomarker of poor prognosis.

Hepatocellular carcinoma (HCC) occurs mainly in the liver associated with chronic hepatitis and hepatic cirrhosis as a result of prolonged viral infection. Transforming growth factor-beta (TGF-beta) induces the fibrosis in hepatic cirrhosis, although it is also an inhibitor of hepatocyte proliferation. To understand the role of TGF-beta signaling in HCC progression, we analyzed gene expression in HCC cells in relation to TGF-beta signaling using a two-way clustering algorithm. By the analysis, five HCC cell lines were classified into two groups according to their metastatic capacity. TGF-beta receptor II (TGFBR2) was downregulated in metastatic cells, which did not show a response to TGF-beta. Immunohistochemistry demonstrated clear membrane distribution of TGFBR2 in noncancerous hepatocytes, whereas reduced TGFBR2 expression was observed in 34 of 136 HCCs. In clinical cases, reduced TGFBR2 expression correlated with larger tumor size (P

Human hepatocellular carcinoma is recognized as a good model for multistage carcinogenesis, as the malignant steps from chronic liver disease through to advanced human hepatocellular carcinoma are relatively clear. We address the activation of different molecular pathways during hepatocarcinogenesis that is especially useful in the diagnosis of pathological multistage human hepatocellular carcinoma. In chronic liver disease, the gene-expression signature as well as the degree of liver fibrosis could help us to predict the development of human hepatocellular carcinoma or survival outcome after treatment for human hepatocellular carcinoma. Several genes, such as HSP70, CAP2 and GPC3, have been identified as potential biomarkers for early human hepatocellular carcinoma. Classical oncogenes or tumor suppressor genes, such as beta-catenin and p53, are mutated during the progression from early to advanced human hepatocellular carcinoma. Also, the presence of hepatoblastic feature like CK19 in advanced human hepatocellular carcinoma can be used as a predictor of aggressive human hepatocellular carcinoma. Although many advances have been made in the diagnosis of multistage hepatocarcinogenesis, we still need further useful markers to more precisely evaluate each step of hepatocarcinogenesis for better treatment choices, and that will promote future molecular-targeted therapy.

Background: Hepatocellular carcinoma (HCC) is one of the most common and most frequently lethal cancers worldwide. Although many advances have been made in the analysis of multistage hepatocarcinogenesis, we still lack information to guide adequate clinical management options for HCC. A large number of genetic alterations occur during hepatocarcinogenesis, and many genetic studies have indicated that one of the most frequently mutated oncogenes found in HCC is β-catenin. Summary: Molecular subclassification of HCC based on gene expression signatures has identified a typical hepatocyte-like subclass of HCC harboring β-catenin mutations; this subclass is characterized by better histological differentiation and a less aggressive nature. We previously identified overexpression of the leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), also known as GPR49, in HCC with β-catenin mutations. LGR5 has been indicated as one of the downstream target genes of the Wnt signaling pathway; however, the functional role of LGR5 in cancer is largely unknown. We demonstrated that HCC cells transfected with LGR5 exhibited higher colony forming activity and were more resistant to a cytotoxic drug than the control HCC cells were. Overexpression of LGR5 also retarded cell migration. LGR5-transfected HCC cells formed nodule-type tumors in the livers of immunodeficient mice, whereas control cells formed more invasive tumors. Results of our recent research suggest that aberrant expression of LGR5 could regulate the epithelial cell phenotype and promotes HCC cell survival. HCC cells overexpressing LGR5 seem to represent a typical phenotype of a less aggressive HCC. Key messages: Recent efforts on the molecular classification of HCC have led us to new strategies for dealing with HCC. These specific signatures may predict the risk of recurrence or the patient survival rate, which affect the outlook and may suggest treatment strategies for HCC patients.

Pancreatic ductal adenocarcinoma (PDA) is a highly aggressive and often lethal malignant tumor. Several studies have shown that epithelial-mesenchymal transition (EMT) is frequently observed in clinical samples of PDA and is related to high metastatic rates and poor outcomes. To identify candidate molecules regulating EMT in PDA, we previously used cDNA microarray analysis and identified integrin β4 (ITGB4) as one of the genes upregulated in high-EMT xenografts derived from PDA patients. The aim of the current study was to clarify the clinicopathological and functional significance of ITGB4 overexpression in PDA. ITGB4 upregulation in high-EMT xenografts was confirmed by immunohistochemistry. Immunohistochemical analyses of 134 surgically resected PDA cases revealed intratumoral heterogeneity with respect to ITGB4 expression and showed that cancer cells undergoing EMT often display strong diffuse ITGB4 expression. High levels of ITGB4 expression were significantly correlated with the hallmarks of EMT (solitary cell infiltration, reduced E-cadherin expression, and increased vimentin expression), with high tumor grade, and with the presence of lymph node metastasis, and showed an independent prognostic effect. Immunocytochemical analyses of PDA cell lines revealed that localization of ITGB4 changed from regions of cell-cell contact to diffuse cytoplasm and cell edges with occasional localization in filopodia during EMT. Knockdown of ITGB4 reduced the migratory and invasive ability of PDA cells. Overexpression of ITGB4 promoted cell scattering and cell motility in combination with downregulation of E-cadherin and upregulation of vimentin expression. In conclusion, we elucidated the prognostic and clinicopathological significance of ITGB4 overexpression in PDA and also the potential role for ITGB4 in the regulation of cancer invasion and EMT.

(Cancer Sci 2010; 101: 666–672) Overexpression of “stemness gene”Bmi-1 has been identified in some solid tumors. We investigated Bmi-1 expression in hepatocellular carcinoma (HCC) and ATP-binding cassette transporter B1 (ABCB1) as a new potential target for Bmi-1. Bmi-1 was highly expressed in HCC cell lines and the most well differentiated cell line, KIM-1, showed the highest expression. Immunohistochemical, immunocytochemical, and immunoelectron microscopic analysis showed the Bmi-1 protein as having a high intensity of small dots within the nucleus which reflected concentrated sites of Bmi-1 repressive activity. Clear “dot-pattern” staining was observed in 24 of 37 (65%) well differentiated HCC (including 13 of 21 early nodules [62%]), in 32 of 71 (45%) moderately differentiated HCC, and 7 of 14 (50%) poorly differentiated HCC. A similar expression was not observed in non-cancerous background regions. High Bmi-1 expression was observed in the early and well differentiated HCC. Furthermore, overexpression and suppression of Bmi-1 was followed by a respective increase and decrease in ABCB1 expression. As with Bmi-1, high ABCB1 expression was also observed in the early and well differentiated HCC. A strong correlation between ABCB1 and Bmi-1 mRNA expression was seen in HCC cell lines and clinical samples (Pearson’s correlation coefficient 0.95 and 0.90, respectively). The Bmi-1 gene is upregulated in HCC, and in particular is highly expressed in early and well differentiated HCC. The fact that this expression correlated with that of ABCB1 suggests a new regulation target for Bmi-1, and gives new insight into early hepatocarcinogenesis mechanisms and potential targets for future HCC treatment.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors. HCC occurs mainly in chronically diseased livers, e.g. following hepatitis B and C infection. These high-risk patients are closely followed up, and increasing numbers of small equivocal lesions are detected by imaging diagnosis. They are now widely recognized as precursor or early-stage HCCs and are classified as dysplastic nodule or early HCC. These lesions lack typical imaging and histology of ordinary HCC and do not show elevated serum markers of α-fetoprotein and PIVKA-II, for example. Molecular analysis of these lesions would help to develop molecular markers for objective histological diagnosis of early HCC and possibly new serum markers for early detection of HCC. It has been reported that HSP70, CAP2, glypican 3 and glutamine synthetase could serve as molecular markers for early HCC. Further analysis is expected to evaluate their usefulness in routine pathological diagnosis including biopsy diagnosis and also as serum markers for early detection of HCC.

Pancreatic cancer is a highly aggressive and lethal malignant tumor. Recent reports have shown that the epithelial-mesenchymal transition (EMT) plays an essential role in cancer invasion and metastasis. We previously reported that EMT was frequently observed in pancreatic ductal adenocarcinoma (PDA) and was related to a high metastatic rate and a poor outcome in patients with PDA (Masugi et al. Hum Pathol. 2010). Further, using cDNA microarray analyses, we identified integrin β4 (ITGB4) as one of the EMT signature genes in 12 patient-derived PDA xenografts. The aim of the current study was to clarify the clinicopathological and functional significance of ITGB4 in PDA. ITGB4 up-regulation in xenografts with high EMT was confirmed by immunohistochemistry. Immunohistochemical analyses of 134 resected PDAs revealed intratumoral heterogeneity with respect to ITGB4 expression and showed that cancer cells undergoing EMT often display diffuse and intense expression of ITGB4. High levels of ITGB4 expression were significantly correlated with the histological hallmarks of EMT (solitary cell infiltration, reduced E-cadherin expression and increased vimentin expression), the grade, and the presence of lymph node metastasis, and showed an independent prognostic effect. Immunocytochemical analyses of PDA cell lines revealed that the distribution of ITGB4 changed from regions of cell-cell contact to a diffuse presence in cytoplasm with occasional localization in filopodia during EMT. Knockdown of ITGB4 by small interfering RNA reduced the migratory and invasive ability of PDA cells. Overexpression of ITGB4 promoted cell scattering and cell motility in combination with down-regulation of E-cadherin and up-regulation of vimentin expression. In conclusion, our study demonstrated the prognostic significance of ITGB4 overexpression in PDA. Moreover, we also identified a potential role for ITGB4 in the regulation of cancer invasion and EMT. Citation Format: Yohei Masugi, Ken Yamazaki, Katsura Emoto, Kathryn Effendi, Minoru Kitago, Osamu Itano, Yuko Kitagawa, Michiie Sakamoto. Up-regulation of integrin β4 promotes epithelial-mesenchymal transition and is a novel prognostic marker in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1146. doi:10.1158/1538-7445.AM2014-1146

Primary cilia are microtubule-based organelles that protrude from basal bodies and are involved in cell differentiation, sensory functions, and planar cell polarity. Especially, primary cilia have become popular as the place for signal transduction pathways including sonic hedgehog, Wnt, and platelet-derived growth factor pathways. Although there are many studies examining the roles of primary cilia in the fields of embryology and physiology, few such studies have been carried out in the field of oncology, and the role of primary cilia in cancer cells is poorly understood especially in human samples. In this study, we identified primary cilia by immunofluorescence analysis in which primary cilia were visualized as green rods labeled with anti-acetylated α-tubulin adjacent to basal bodies detected as red dots labeled with anti-γ-tubulin. Primary cilia were found in human pancreatic cancer cell lines (PANC-1 and CFPAC-1) and the number of them increased in the condition of serum starvation. As for clinical samples, primary cilia were found in cancer cells in 25 of 100 pancreatic ductal carcinoma patients [1-109 (median 8) primary cilia / 3 field pictures]. Most primary cilia in cancer tissue were observed in areas showing well differentiated glandular structures in clinical samples. Patients whose cancers were primary cilia positive had a higher frequency of lymph node metastasis than those whose cancers were primary cilia negative (p = 0.016). Univariate analysis demonstrated that tumor size (p = 0.009), tumor grade (p = 0.001), lymph node metastasis (p = 0.008), and the presence of primary cilia (p = 0.002) correlated with overall survival. Multivariate analysis found that tumor grade (p < 0.001) and the presence of primary cilia (p = 0.001) were independent prognostic indicators. In summary, we showed that pancreatic cancer cells can form primary cilia and that the presence of primary cilia is significantly associated with the prognosis of pancreatic ductal adenocarcinoma. The presence of primary cilia has the potential to become a new diagnostic tool and therapeutic target. Citation Format: Katsura Emoto, Yohei Masugi, Ken Yamazaki, Kathryn Effendi, Hanako Tsujikawa, Minoru Kitago, Osamu Itano, Yuko Kitagawa, Michiie Sakamoto. Presence of primary cilia in cancer cells correlates with prognosis of pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 179. doi:10.1158/1538-7445.AM2014-179

Pancreatic cancer has one of the worst survival rates among all cancers. Features like epithelial-mesenchymal transition (EMT) are often seen in pancreatic tumors and correlate with poor prognosis. The purpose of this study was to identify factors involved in EMT in pancreatic cancer that may serve as prognostic indicators. Gene-expression profiling identified overexpression of SMAD3 in pancreatic cancer with high-grade solitary cell infiltration, one of the EMT-like features. SMAD3 expression was then evaluated through immunohistochemical analysis using clinical specimens. SMAD3 was accumulated in the nuclei of tumor cells (positive rate ranging from 0% to 75%), but was not detected in most of the epithelial cells in the pancreatic duct. Upregulated SMAD3 (≥15% positive in nuclei of tumor cells) correlated with malignant characteristics, such as higher tumor grade (P = 0.001) and lymph node metastasis (P Citation Format: Ken Yamazaki, Yohei Masugi, Kathryn Effendi, Michiie Sakamoto. SMAD3 upregulation is a novel biomarker of poor prognosis and epithelial-mesenchymal transition in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 417. doi:10.1158/1538-7445.AM2013-417