Published papers

© 2018 The Japan Society of Hepatology Aim: Hepatic fibrosis is the most important factor for estimating the prognosis of patients with non-alcoholic fatty liver disease (NAFLD). A novel non-invasive scoring system, the FM-fibro index, showed high accuracy in a pilot study. The purpose of this study was to validate the efficacy of the FM-fibro index in a multicenter cohort. Methods: Among 18 institutions, we analyzed 400 Japanese patients with biopsy-proven NAFLD. We evaluated the accuracies of the FM-fibro index, CA-fibro index, and European Liver Fibrosis (ELF) panel by area under the receiver operator characteristics curves (AUROC). The FM-fibro index includes three formulas for type IV collagen 7S, hyaluronic acid, and vascular cell adhesion molecule-1. Results: Among 400 patients, 205 were women, and the median age was 56 years. The histological distribution of Matteoni types 1, 2, 3, and 4 was 11, 40, 15, and 334, and the distribution of hepatic fibrosis stages 0 to 4 was 67, 183, 55, 63, and 32, respectively. The AUROCs of the FM-fibro index, CA-fibro index, and ELF panel for non-alcoholic steatohepatitis (NASH)-related fibrosis were 0.7178/0.7095/0.7065, 0.7093, and 0.7245, respectively. The sensitivity and specificity of the FM-fibro index for predicting NASH-related fibrosis was 0.5359/0.5210/0.4641 and 0.8333/0.8182/0.8788, respectively. The accuracy of the FM-fibro index was not significantly different from that of the CA-fibro index or the ELF panel. Conclusions: The FM-fibro index can predict NASH-related fibrosis with sufficient accuracy compared with previous scoring systems. Further analyses that verify the accuracy of the FM-fibro index to distinguish significant or advanced fibrosis in patients with NAFLD are awaited. (UMIN-CTR: UMIN000018158).

© 2018, The Author(s). CD26, a 110-kDa transmembrane glycoprotein that is expressed on several tumor cells including malignant lymphoma, has been implicated in tumorigenesis: however, little is known regarding its role in multiple myeloma (MM). Recently, we identified CD26 expression on human osteoclasts (OCs) and demonstrated that humanized IgG1 monoclonal antibody targeting CD26, huCD26mAb, inhibits human OC differentiation. Herein, we show that CD26 expression was present on plasma cells in the bone marrow tissues of MM patients. In vitro immunostaining studies revealed that although CD26 expression was low or absent on MM cell lines cultured alone, it was intensely and uniformly expressed on MM cell lines co-cultured with OCs. The augmented CD26 expression in MM cells was exploited to enhance anti-MM efficacy of huCD26mAb via a substantial increase in antibody-dependent cytotoxicity (ADCC) but not complement-dependent cytotoxicity (CDC). Moreover, huCD26mAb in combination with novel agents synergistically enhanced huCD26mAb induced ADCC activity against CD26+ MM cells compared with each agent alone. huCD26mAb additionally reduced the ratio of the side population (SP) fraction in CD26+ MM cells by ADCC. Finally, huCD26mAb significantly reduced the MM tumor burden and OC formation in vivo. These results suggest that CD26 is a potential target molecule in MM and that huCD26mAb could act as a therapeutic agent.

© 2018 by the American Association for the Study of Liver Diseases. Primary liver carcinomas with both hepatocytic and cholangiocytic differentiation have been referred to as “combined (or mixed) hepatocellular-cholangiocarcinoma.” These tumors, although described over 100 years ago, have attracted greater attention recently because of interest in possible stem cell origin and perhaps because of greater frequency and clinical recognition. Currently, because of a lack of common terminology in the literature, effective treatment and predictable outcome data have been challenging to accrue. This article represents a consensus document from an international community of pathologists, radiologists, and clinicians who have studied and reported on these tumors and recommends a working terminology for diagnostic and research approaches for further study and evaluation. Conclusion: It is recommended that diagnosis is based on routine histopathology with hematoxylin and eosin (H&E); immunostains are supportive, but not essential for diagnosis. (Hepatology 2018;68:113-126).

© 2018 The Author(s). Background: Since the concept of intraductal tubulopapillary neoplasm (ITPN) was introduced in the current World Health Organization classification of tumors, the number of reports of ITPN occurrence has increased gradually. However, ITPN is usually located in the main pancreatic duct, with few reports of a branch duct ITPN. As a result, imaging protocols for the diagnosis of a branch duct ITPN have not been established. Case presentation: We report a case of a concurrent presentation of a branch duct ITPN and intraductal papillary mucinous neoplasm (IPMN) in the head of the pancreas, with a superior mesenteric artery (SMA) aneurysm. Initially, the cystic masses in the pancreatic head were diagnosed as branch duct IPMNs, with treatment consisting of a pylorus-preserving pancreaticoduodenectomy, in combination with an aneurysmectomy performed for treatment of the SMA aneurysm. Pathological examination confirmed these cysts were a combination of branch-type ITPN and IPMN. The patient recovered from the treatment without complication, with no evidence of recurrence over a period of 34 months post-surgery. Conclusion: This case report of a synchronous presentation of an ITPN and IPMN indicates the difficulty in differentiating these two types of neoplasms in the branch duct of the pancreatic head.

© 2018, ISAROB. Advances in whole-slide scanning have led to research on digital pathology, including monitor-based diagnosis, feature quantification, and computer-aided diagnosis. The color variations due to the staining process and scanning device are a serious issue that should be solved in whole-slide imaging applications, and methods for color correction have been studied. Nevertheless, the effectiveness of color-correction methods in the quantitative analysis of histopathological images acquired by different whole-slide scanners (WSSs) has not been confirmed. In this work, several liver tissue samples were scanned by different WSSs, and a color-correction method for whole-slide images was applied to the digitized tissue specimens. A set of histological features were extracted from the histopathological images, both without and with color correction, to analyze the effectiveness of the color-correction method in feature quantification and cancer identification.

© 2018 Wolters Kluwer Health, Inc. All rights reserved. Objectives The aim of this study was to determine whether computer-assisted digital analysis and acoustic radiation force impulse (ARFI) imaging were useful for assessing pancreatic fibrosis, and if ARFI imaging predicted postoperative pancreatic fistula (POPF). Methods Seventy-eight patients scheduled to undergo pancreatic resection were enrolled. Shear wave velocity (SWV) at the pancreatic neck was measured preoperatively using ARFI imaging. Pancreatic tissue components on a whole slide image were quantified using an automatic image processing software. The relationship between SWV, fibrotic tissue content, and POPF incidence and clinical severity was analyzed. Results The median collagen fiber, fatty tissue, and acinar cell contents were 11.6%, 8.5%, and 61.3%, respectively. Unlike fatty tissue, collagen fiber content and acinar cells were correlated with SWV (ρ = 0.440, P < 0.001 and ρ =-0.428, P < 0.001, respectively). Although collagen fiber content and SWV were associated with the overall incidence of POPF (P = 0.004 and 0.001, respectively), collagen fiber content and SWV had no statistical correlation with clinically relevant POPF (P = 0.268 and 0.052, respectively). Conclusions We objectively quantified the pancreatic tissue components using an automatic image processing software. Shear wave velocity was significantly related to collagen fiber content and suggests that ARFI imaging can be useful for evaluating pancreatic fibrosis.

© 2018 Japanese Dermatological Association Activation of the Hedgehog (HH) signaling pathway plays a critical role in the development of basal cell carcinoma (BCC). HH signaling activity is produced by nuclear translocation of transcription factors, glioma-associated oncogene homolog (GLI). Among three GLI subfamilies, GLI1 is the only full-length transcriptional activator, and its nuclear localization is recognized as a signature event in HH signaling activation. However, limited published work has investigated the nuclear staining of GLI1 protein in human tumor tissue samples by immunohistochemical analysis. In this study, we performed immunohistochemical staining of GLI1 in 382 cases of cutaneous epithelial tumors, including 196 BCC cases, using rabbit monoclonal antihuman GLI1 antibody (C68H3). As a result, 98.2% cases of BCC showed a diffuse and strong nuclear staining pattern regardless of the histological subtype. Positive staining was mainly restricted to the tumor nests, while the overlying epidermis was negative suggesting specificity of the antibody. In further analysis of other cutaneous epithelial tumors, 100% (4/4) cases of trichoblastoma, 15.1% (5/33) Bowen's disease, 3.5% (1/28) actinic keratosis and 12.5% (4/32) squamous cell carcinoma showed the nuclear staining pattern of GLI1. This suggested that HH signaling is also dysregulated in some other cutaneous malignant tumors. In conclusion, the C68H3 antibody is a useful tool for revealing activation of HH signaling in immunohistochemical analysis.

© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Background and Aims: Small hypovascular hepatocellular carcinoma (HCC) ≤2 cm is biologically less aggressive than hypervascular one, however, the optimal treatment is still undetermined. The efficacy of surgical resection (SR), radiofrequency ablation (RFA) and percutaneous ethanol injection (PEI) was evaluated. Methods: The 853 (SR, 176; RFA, 491; PEI, 186) patients were enrolled who met Child-Pugh A/B, single hypovascular HCC ≤2 cm pathologically proven, available tumour differentiation and absence of macrovascular invasion and extrahepatic metastasis. Overall and recurrence-free survivals were compared in original and a propensity score weighted pseudo-population with 732 patients. Results: The median follow-up time and tumour size were 2.8 years and 1.47 cm respectively. In original population, multivariate Cox regression showed no significant difference for overall survival among three groups. In pseudo-population, Cox regression also revealed no significant difference for overall survival among them, although SR (HR, 0.56; 95% CI, 0.36-0.86) and RFA (HR, 0.75; 95% CI, 0.57-1.00) groups had significantly lower recurrence than PEI group. The overall survival rates at 3 and 5 years for the SR, RFA and PEI groups were 94%/70%, 90%/75% and 94%/73% respectively. Corresponding recurrence-free survival rates were 64%/54%, 59%/41% 48%/33% respectively. Subgroup analysis revealed no significant survival benefit of SR compared with non-SR. No treatment-related death occurred. Conclusions: For patients with single hypovascular HCC ≤2 cm, no significant difference for overall survival was first identified among 3 treatment groups. The SR or RFA could be recommended, and PEI would be alternative to RFA.

© 2018 by the American Association for the Study of Liver Diseases. Immune cells constitute an important element of tumor tissue. Accumulating evidence indicates their clinicopathological significance in predicting prognosis and therapeutic efficacy. Nonetheless, the combinations of immune cells forming the immune microenvironment and their association with histological findings remain largely unknown. Moreover, it is unclear which immune cells or immune microenvironments are the most prognostically significant. Here, we comprehensively analyzed the immune microenvironment and its intratumor heterogeneity in 919 regions of 158 hepatocellular carcinomas (HCCs), and the results were compared with the corresponding histological and prognostic data. Consequently, we classified the immune microenvironment of HCC into three distinct immunosubtypes: Immune-high, Immune-mid, and Immune-low. The Immune-high subtype was characterized by increased B-/plasma-cell and T cell infiltration, and the Immune-high subtype and B-cell infiltration were identified as independent positive prognostic factors. Varying degrees of intratumor heterogeneity of the immune microenvironment were observed, some of which reflected the multistep nature of HCC carcinogenesis. However, the predominant pattern of immunosubtype and immune cell infiltration of each tumor was prognostically important. Of note, the Immune-high subtype was associated with poorly differentiated HCC, cytokeratin 19 (CK19) + , and/or Sal-like protein 4 (SALL4) + high-grade HCC, and Hoshida's S1/Boyault's G2 subclasses. Furthermore, patients with high-grade HCC of the predominant Immune-high subtype had significantly better prognosis. These results provide a rationale for evaluating the immune microenvironment in addition to the usual histological/molecular classification of HCC. Conclusion: The immune microenvironment of HCC can be classified into three immunosubtypes (Immune-high, Immune-mid, and Immune-low) with additional prognostic impact on histological and molecular classification of HCC. (Hepatology 2018).

© 2018 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd Epithelial–mesenchymal transition (EMT) promotes invasion and metastasis of pancreatic ductal adenocarcinoma (PDAC). However, the importance of its reverse process, mesenchymal–epithelial transition (MET), for PDAC remains unclear. We aimed to characterize the histological finding “focal differentiation” in PDAC at perineural invasion sites in the context of MET and to investigate the role of Schwann cells in inducing tumor MET. Tumor differentiation and immunohistochemical expressions of E-cadherin, SMAD3, and vimentin at perineural invasion sites were examined in 168 PDAC tissues. Four PDAC cell lines were co-cultured with Schwann cells to investigate cell morphology, motility, or EMT-related markers using immunocytochemistry and quantitative PCR. Of 168 tumors, 124 (74%) showed focal differentiation with enhanced E-cadherin membrane expression (P < 0.001) and decreased nuclear accumulation of SMAD3 (P < 0.001). Among 115 PDACs harboring grade 1/2 tumor, tumors with focal differentiation showed worse survival compared to those without focal differentiation (P = 0.019). PDAC cells co-cultured with Schwann cells demonstrated a sheet-like appearance, increased E-cadherin expression, decreased expressions of SMAD3 and vimentin, and reduced cell motility. In conclusion, MET-like change is induced by Schwann cells, suggesting that Schwann cells contribute to PDAC colonization in pancreatic nerves through activating the MET machinery inside tumor cells in the pancreatic tumor microenvironment.

© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. The aim of this study was to develop a new methodology that is suitable for DNA methylation diagnostics and to demonstrate its clinical applicability. We developed a new anion-exchange column for high-performance liquid chromatography (HPLC) with electrostatic and hydrophobic properties. Both cytosine and thymine, corresponding to methylated and unmethylated cytosine after bisulfite modification, respectively, are captured by electrostatic interaction and then discriminated from each other by their hydrophobic interactions. The DNA methylation levels of synthetic DNA were quantified accurately and reproducibly within 10 minutes without time-consuming pretreatment of PCR products, and the measured values were unaffected by the distribution of methylated CpG within the synthetic DNA fragments. When the DNA methylation status of the FAM150A gene, a marker of the CpG island methylator phenotype specific to clear cell renal cell carcinoma (ccRCC), was examined in 98 patients with ccRCC, bulk specimens of tumorous tissue including cancer cells showing DNA methylation of the FAM150A gene were easily identifiable by simply viewing the differentiated chromatograms, even when the cancer cell content was low. Sixteen ccRCC showing DNA methylation more frequently exhibited clinicopathological parameters reflecting tumor aggressiveness (ie, a larger diameter, higher histological grade, vascular involvement, renal vein tumor thrombi, infiltrating growth, tumor necrosis, renal pelvis invasion and higher pathological TNM stage), and had significantly lower recurrence-free and overall survival rates. These data indicate that HPLC analysis using this newly developed anion-exchange column could be a powerful tool for DNA methylation diagnostics, including prognostication of patients with cancers, in a clinical setting.

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.

We classify the combinations of parameters which lead three generations of quarks and leptons in the framework of magnetized twisted orbifolds on T-2/Z(2), T-2/Z(3), T-2/Z(4) and T-2/Z(6) with allowing nonzero discretized Wilson line phases and Scherk-Schwarz phases. We also analyze two actual examples with nonzero phases leading to one-pair Higgs and five-pair Higgses and discuss the difference from the results without nonzero phases studied previously. (Note: this article is registered under preprint number: arXiv:1501.02787 [hep-ph].) (C) 2015 The Authors. Published by Elsevier B.V.

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 beta 4 (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.