Papers - Sakamoto, Michiie

Effectiveness of color correction on the quantitative analysis of histopathological images acquired by different whole-slide scanners

Aziz M., Nakamura T., Yamaguchi M., Kiyuna T., Yamashita Y., Abe T., Hashiguchi A., Sakamoto M.

Artificial Life and Robotics (Artificial Life and Robotics)  24 ( 1 ) 28 - 37 2019.03

ISSN  14335298

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© 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.

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Accuracy of non-invasive scoring systems for diagnosing non-alcoholic steatohepatitis-related fibrosis: Multicenter validation study

Itoh Y., Seko Y., Shima T., Nakajima T., Mizuno K., Kawamura Y., Akuta N., Ito K., Kawanaka M., Hiramatsu A., Sakamoto M., Harada K., Goto Y., Nakayama T., Kumada H., Okanoue T.

Hepatology Research (Hepatology Research)  48 ( 13 ) 1099 - 1107 2018.12

ISSN  13866346

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© 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).

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CD26 is a potential therapeutic target by humanized monoclonal antibody for the treatment of multiple myeloma

Nishida H., Hayashi M., Morimoto C., Sakamoto M., Yamada T.

Blood Cancer Journal (Blood Cancer Journal)  8 ( 11 )  2018.11

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© 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.

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Evaluation of pancreatic fibrosis with acoustic radiation force impulse imaging and automated quantification of pancreatic tissue components

Fujita Y., Kitago M., Abe T., Itano O., Shinoda M., Abe Y., Yagi H., Hibi T., Ishii M., Nakano Y., Okuma K., Hashimoto M., Takeuchi A., Masugi Y., Jinzaki M., Sakamoto M., Kitagawa Y.

Pancreas (Pancreas)  47 ( 10 ) 1277 - 1282 2018.11

ISSN  08853177

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© 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.

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Immunohistochemical visualization of the signature of activated Hedgehog signaling pathway in cutaneous epithelial tumors

Tanese K., Emoto K., Kubota N., Fukuma M., Sakamoto M.

Journal of Dermatology (Journal of Dermatology)  45 ( 10 ) 1181 - 1186 2018.10

ISSN  03852407

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© 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.

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Landscape of immune microenvironment in hepatocellular carcinoma and its additional impact on histological and molecular classification

Kurebayashi Y., Ojima H., Tsujikawa H., Kubota N., Maehara J., Abe Y., Kitago M., Shinoda M., Kitagawa Y., Sakamoto M.

Hepatology (Hepatology)  68 ( 3 ) 1025 - 1041 2018.09

ISSN  02709139

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© 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).

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cHCC-CCA: Consensus terminology for primary liver carcinomas with both hepatocytic and cholangiocytic differentation

Brunt E., Aishima S., Clavien P., Fowler K., Goodman Z., Gores G., Gouw A., Kagen A., Klimstra D., Komuta M., Kondo F., Miksad R., Nakano M., Nakanuma Y., Ng I., Paradis V., Nyun Park Y., Quaglia A., Roncalli M., Roskams T., Sakamoto M., Saxena R., Sempoux C., Sirlin C., Stueck A., Thung S., Tsui W., Wang X., Wee A., Yano H., Yeh M., Zen Y., Zucman-Rossi J., Theise N.

Hepatology (Hepatology)  68 ( 1 ) 113 - 126 2018.07

ISSN  02709139

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© 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).

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Novel method for DNA methylation analysis using high-performance liquid chromatography and its clinical application

Yotani T., Yamada Y., Arai E., Tian Y., Gotoh M., Komiyama M., Fujimoto H., Sakamoto M., Kanai Y.

Cancer Science (Cancer Science)  109 ( 5 ) 1690 - 1700 2018.05

ISSN  13479032

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© 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.

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Concurrent presentation of an intraductal tubulopapillary neoplasm and intraductal papillary mucinous neoplasm in the branch duct of the pancreas, with a superior mesenteric artery aneurysm: A case report

Inomata K., Kitago M., Obara H., Fujii-Nishimura Y., Shinoda M., Yagi H., Abe Y., Hibi T., Matsubara K., Oshima G., Sekimoto Y., Inoue M., Itano O., Sakamoto M., Kitagawa Y.

World Journal of Surgical Oncology (World Journal of Surgical Oncology)  16 ( 1 )  2018.04

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© 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.

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Mesenchymal–epithelial transition of pancreatic cancer cells at perineural invasion sites is induced by Schwann cells

Fujii-Nishimura Y., Yamazaki K., Masugi Y., Douguchi J., Kurebayashi Y., Kubota N., Ojima H., Kitago M., Shinoda M., Hashiguchi A., Sakamoto M.

Pathology International (Pathology International)  68 ( 4 ) 214 - 223 2018.04

ISSN  13205463

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© 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.

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Impact of resection and ablation for single hypovascular hepatocellular carcinoma ≤2 cm analysed with propensity score weighting

Takayasu K., Arii S., Sakamoto M., Matsuyama Y., Kudo M., Kaneko S., Nakashima O., Kadoya M., Izumi N., Takayama T., Ku Y., Kumada T., Kubo S., Kokudo T., Hagiwara Y., Kokudo N.

Liver International (Liver International)  38 ( 3 ) 484 - 493 2018.03

ISSN  14783223

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© 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.

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Overexpression of adenylate cyclase-associated protein 2 is a novel prognostic marker in malignant melanoma

Masugi, Y., Tanese, K., Emoto, K., Yamazaki, K., Effendi, K., Funakoshi, T., Mori, M. and Sakamoto, M.

Pathol Int  2015.09

ISSN  1440-1827

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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.

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Classification of three-generation models on magnetized orbifolds

Abe, T., Fujimoto, Y., Kobayashi, T., Miura, T., Nishiwaki, K., Sakamoto, M. and Tatsuta, Y.

Nuclear Physics B 894   374 - 406 2015.05

ISSN  0550-3213

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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.

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Upregulation of integrin beta 4 promotes epithelial-mesenchymal transition and is a novel prognostic marker in pancreatic ductal adenocarcinoma

Masugi, Y., Yamazaki, K., Emoto, K., Effendi, K., Tsujikawa, H., Kitago, M., Itano, O., Kitagawa, Y. and Sakamoto, M.

Laboratory Investigation 95 ( 3 ) 308 - 319 2015.03

ISSN  0023-6837

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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.

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OATP1B3 expression is strongly associated with Wnt/beta-catenin signalling and represents the transporter of gadoxetic acid in hepatocellular carcinoma

Ueno, A., Masugi, Y., Yamazaki, K., Komuta, M., Effendi, K., Tanami, Y., Tsujikawa, H., Tanimoto, A., Okuda, S., Itano, O., Kitagawa, Y., Kuribayashi, S. and Sakamoto, M.

Journal of Hepatology 61 ( 5 ) 1080 - 1087 2014.11

ISSN  0168-8278

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Background & Aims: In the current era of emerging molecular targeted drugs, it is necessary to identify before treatment the specific subclass to which a tumour belongs. Gadoxetic acid is a liver-specific contrast agent that is preferentially taken up by hepatocytes. Therefore, gadoxetic acid-enhanced magnetic resonance imaging (EOB-MRI) should provide precise molecular information about hepatocellular carcinomas (HCCs). The aim of this study was to investigate the transporters of gadoxetic acid in HCC comprehensively and to analyse the molecular regulatory mechanism of such transporters.

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Quantification of collagen and elastic fibers using whole-slide images of liver biopsy specimens

Abe, T., Hashiguchi, A., Yamazaki, K., Ebinuma, H., Saito, H., Kumada, H., Izumi, N., Masaki, N. and Sakamoto, M.

Pathology International 63 ( 6 ) 305 - 310 2013.06

ISSN  1320-5463

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Histological evaluation of fibrosis after a liver biopsy is crucial for evaluating the pathology of patients with chronic liver disease. Previous studies have reported quantitative analyses of fibrosis using images of collagen-stained sections. However, analysis of these studies requires manual selection of the region of interest. In addition, the quantification of elastic fibers is not considered. The present study was conducted in order to measure both the collagen and elastic fiber area ratios using Elastica van Gieson-stained whole-slide images (WSIs) of liver biopsy specimens. High-resolution WSIs provide precise color classification, enabling accurate detection of even fine collagen and elastic fibers. To minimize the influence of pre-existing fibrous tissue, median area ratios of the collagen and elastic fibers were independently calculated from the image tiles of the WSIs. These median area ratios were highly concordant with area ratios after the pre-existing fibrous tissues were manually trimmed from the WSI. Further, these median area ratios were correlated with liver stiffness as measured by transient elastography (collagen: r=0.73 [P<0.01], elastic: r=0.53 [P<0.01]). Our approach to quantifying liver fibrosis will serve as an effective tool to evaluate liver diseases in routine practice.

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Leucine-rich repeat-containing G protein-coupled receptor 5 regulates epithelial cell phenotype and survival of hepatocellular carcinoma cells

Fukuma, M., Tanese, K., Effendi, K., Yamazaki, K., Masugi, Y., Suda, M. and Sakamoto, M.

Experimental Cell Research 319 ( 3 ) 113 - 121 2013.02

ISSN  0014-4827

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The leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), also known as GPR49, is a seven-transmembrane receptor that is expressed in stem cells of the intestinal crypts and hair follicles of mice. LGR5 is overexpressed in some types of human cancer, and is one of the target genes of the Wnt signaling pathway. To explore the function of LGR5 in cancer cells, stable hepatocellular carcinoma (HCC) cell lines expressing FLAG-tagged LGR5 were established. Overexpression of LGR5 resulted in changes in cell shape from an extended flat (mesenchymal) phenotype to a round aggregated (stem cell-like) phenotype. Cells transfected with LGR5 showed higher colony forming activity, and were more resistant to a cytotoxic drug than cells transfected with empty vector. Overexpression of LGR5 inhibited cell motility. LGR5-transfected cells formed nodule type tumors in the livers of immunodeficient mice, whereas empty vector-transfected cells formed more invasive tumors. Down-regulation of LGR5 changed the morphology of HCC cells from the aggregated phenotype to an extended spindle phenotype, and cell motility was increased. This is the first study reporting the functional role of LGR5 in the biology of HCC cells, and the results suggest that aberrant expression of LGR5 regulates epithelial cell phenotype and survival. (c) 2012 Elsevier Inc. All rights reserved.

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A serum "sweet-doughnut" protein facilitates fibrosis evaluation and therapy assessment in patients with viral hepatitis

Kuno, A., Ikehara, Y., Tanaka, Y., Ito, K., Matsuda, A., Sekiya, S., Hige, S., Sakamoto, M., Kage, M., Mizokami, M. and Narimatsu, H.

Scientific Reports 3 2013.01

ISSN  2045-2322

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Although liver fibrosis reflects disease severity in chronic hepatitis patients, there has been no simple and accurate system to evaluate the therapeutic effect based on fibrosis. We developed a glycan-based immunoassay, FastLec-Hepa, to fill this unmet need. FastLec-Hepa automatically detects unique fibrosis-related glyco-alteration in serum hyperglycosylated Mac-2 binding protein within 20 min. The serum FastLec-Hepa counts increased with advancing fibrosis and illustrated significant differences in medians between all fibrosis stages. FastLec-Hepa is sufficiently sensitive and quantitative to evaluate the effects of PEG-interferon-alpha/ribavirin therapy in a short post-therapeutic interval. The obtained fibrosis progression is equivalent to -0.30 stages/year in patients with sustained virological response, and 0.01 stages/year in relapse/nonresponders. Furthermore, long-term follow-up of the severely affected patients found hepatocellular carcinoma developed in patients after therapy whose FastLec-Hepa counts remained above a designated cutoff value. FastLec-Hepa is the only assay currently available for clinically beneficial therapy evaluation through quantitation of disease severity.

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Involvement of hepatocellular carcinoma biomarker, cyclase-associated protein 2 in zebrafish body development and cancer progression

Effendi, K., Yamazaki, K., Mori, T., Masugi, Y., Makino, S. and Sakamoto, M.

Experimental Cell Research 319 ( 1 ) 35 - 44 2013.01

ISSN  0014-4827

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Cyclase-associated protein 2 (CAP2) is a conserved protein that is found up-regulated in hepatocellular carcinoma (HCC). By using zebrafish, combined with HCC cell lines, we further investigated the role of CAP2. The zebrafish CAP2 sequence was 60% identical to human CAP2 with 77% homology in the C-terminal actin-binding domain, and 58% in the N-terminal cyclase-binding domain. CAP2 expression was observed during zebrafish development and was preferentially expressed in the skeletal muscle and heart. Knockdown using two different morpholinos against CAP2 resulted in a short-body morphant zebrafish phenotype with pericardial edema. CAP2 was observed co-localized with actin in zebrafish skeletal muscle, and in the leading edge of lamellipodium in HCC cell lines. CAP2 silencing resulted in a defect in lamellipodium formation and decreased cell motility in HCC cell lines. Strongly positive expression of CAP2 was observed in 10 of 16 (63%) poorly, 30 of 68 (44%) moderately, and 2 of 21 (10%) well differentiated HCC. CAP2 expression was significantly associated with tumor size, poor differentiation, portal vein invasion, and intrahepatic metastasis. Our results indicate that an important conserved function of CAP2 in higher vertebrates may be associated with the process of skeletal muscle development. CAP2 also played an important role in enhancing cell motility, which may promote a more invasive behavior in the progression of HCC. These findings highlight the link between development and cancer. (C) 2012 Elsevier Inc. All rights reserved.

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Investigation of proNT/NMN secretion from small cell lung carcinoma cells using a mouse xenograft model

K. Wakabayashi-Nakao, K. Maruyama, H. Ishii, K. Muramatsu, K. Hatakeyama, K. Ohshima, S. Ogura, T. Nakajima, K. Yamaguchi and T. Mochizuki

Oncol Rep 28 ( 4 ) 1181 - 6 2012.10

ISSN  1791-2431

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Proneurotensin/neuromedin N (proNT/NMN), the precursor of neurotensin (NT) and neuromedin N (NMN), is produced by cancer tissues derived from the pancreas and colon. NT stimulates tumor growth and proliferation through its receptors; however, little is known about the precursor molecule in cancer tissues. We previously demonstrated that proNT/NMN is secreted from small cell lung carcinoma (SCLC) cell lines in serum-free conditioned medium, but not from non-small cell lung carcinoma (NSCLC) cell lines. It was suggested that this precursor may serve as a tumor marker for SCLC. In this study, we established in vivo xenograft models to evaluate the possibility of proNT/NMN as a specific tumor marker. SBC3 cells, derived from human SCLC, were inoculated into mice, and the proNT/NMN levels in plasma and tumor tissues were detected using specific antibodies. In contrast to control mouse plasma, the proNT/NMN levels in tumor-bearing mice increased as the tumors grew, and the elevated plasma proNT/NMN levels were decreased by tumor resection. Moreover, proNT/NMN was expressed in SBC3 tumors, suggesting that proNT/NMN was secreted into blood from the tumor, and this secretion may be specific to SCLC.

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Ubiquitin-mediated proteasomal degradation of ABC transporters: a new aspect of genetic polymorphisms and clinical impacts

H. Nakagawa, Y. Toyoda, K. Wakabayashi-Nakao, H. Tamaki, M. Osumi and T. Ishikawa

J Pharm Sci 100 ( 9 ) 3602 - 19 2011.09

ISSN  1520-6017

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The interindividual variation in the rate of drug metabolism and disposition has been known for many years. Pharmacogenomics dealing with heredity and response to drugs is a part of science that attempts to explain variability of drug responses and to search for the genetic basis of such variations or differences. Genetic polymorphisms of drug metabolizing enzymes and drug transporters have been found to play a significant role in the patients' responses to medication. Accumulating evidence demonstrates that certain nonsynonymous polymorphisms have great impacts on the protein stability and degradation, as well as the function of drug metabolizing enzymes and transporters. The aim of this review article is to address a new aspect of protein quality control in the endoplasmic reticulum and to present examples regarding the impact of nonsynonymous single-nucleotide polymorphisms on the protein stability of thiopurine S-methyltransferase as well as ATP-binding cassette (ABC) transporters including ABCC4, cystic fibrosis transmembrane conductance regulator (CFTR, ABCC7), ABCC11, and ABCG2. Furthermore, we will discuss the molecular mechanisms underlying posttranslational modifications (intramolecular and intermolecular disulfide bond formation and N-linked glycosylation) and ubiquitin-mediated proteasomal degradation of ABCG2, one of the major drug transporter proteins in humans.

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Nrk, an X-linked protein kinase in the germinal center kinase family, is required for placental development and fetoplacental induction of labor

K. Denda, K. Nakao-Wakabayashi, N. Okamoto, N. Kitamura, J. Y. Ryu, Y. Tagawa, T. Ichisaka, S. Yamanaka and M. Komada

J Biol Chem 286 ( 33 ) 28802 - 10 2011.08

ISSN  1083-351X

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The complete mechanism of labor induction in eutherian mammals remains unclear. Although important roles for the fetus and placenta in triggering labor have been proposed, no gene has been shown to be required in the fetus/placenta for labor induction. Here we show that Nrk, an X-linked gene encoding a Ser/Thr kinase of the germinal center kinase family, is essential in the fetus/placenta for labor in mice. Nrk was specifically expressed in the spongiotrophoblast layer, a fetus-derived region of the placenta, and Nrk disruption caused dysregulated overgrowth of the layer. Due to preferential inactivation of the paternally derived X chromosome in placenta, Nrk heterozygous mutant placentas exhibited a similar defect to that in Nrk-null tissues when the wild-type allele was paternally derived. However, the phenotype was weaker than in Nrk-null placentas due to leaky Nrk expression from the inactivated X chromosome. Crossing of Nrk-null females to wild-type and Nrk-null males, as well as uterine transfer of Nrk-null fetuses to wild-type females, revealed that pregnant mice exhibit a severe defect in delivery when all fetuses/placentas are Nrk-null. In addition, Nrk was not expressed in female reproductive tissues such as the uterus and ovary, as well as the fetal amnion and yolk sac, in pregnant mice. Progesterone and estrogen levels in the maternal circulation and placenta, which control the timing of labor, were unaffected upon Nrk disruption. We thus provide evidence for a novel labor-inducing fetoplacental signal that depends on the X chromosome and possibly arises from the placenta.

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Identification by differential tissue proteome analysis of talin-1 as a novel molecular marker of progression of hepatocellular carcinoma

Kanamori, H., Kawakami, T., Effendi, K., Yamazaki, K., Mori, T., Ebinuma, H., Masugi, Y., Du, W., Nagasaka, K., Ogiwara, A., Kyono, Y., Tanabe, M., Saito, H., Hibi, T. and Sakamoto, M.

Oncology 80 ( 5-6 ) 406 - 15 2011

ISSN  1423-0232

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OBJECTIVE: Hepatocellular carcinoma (HCC) is characterized by a multistage process of tumor progression. This study addressed its molecular features to identify novel protein candidates involved in HCC progression. METHODS: Using liquid chromatography-tandem mass spectrometry, proteomes of 4 early HCCs and 4 non-HCC tissues derived from 2 cases of liver transplant surgery were compared with respect to the separation profiles of their tryptic peptides. Immunohistochemistry was performed on 106 HCC nodules to confirm the results of the proteomic analysis. RESULTS: Statistical analysis of the profiles selected the peptide peaks differentiating HCC from non-HCC. A database search of the tandem mass spectrometry data from those peptide peaks identified 61 proteins, including a cytoskeletal protein, talin-1, as upregulated in HCC. Talin-1 expression levels in HCC nodules were significantly associated with the dedifferentiation of HCC (p = 0.001). A follow-up survey of the examined clinical cases revealed a correlation between talin-1 upregulation and a shorter time to recurrence after resection (p = 0.039), which may be related to the higher rate of portal vein invasion in HCCs with talin-1 up-regulation (p = 0.029). CONCLUSIONS: Proteomic analysis led to identification of talin-1 as a promising HCC marker. Talin-1 upregulation is associated with HCC progression and may serve as a prognostic marker.

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Expression of Keratin 19 Is Related to High Recurrence of Hepatocellular Carcinoma after Radiofrequency Ablation

Tsuchiya, K., Komuta, M., Yasui, Y., Tamaki, N., Hosokawa, T., Ueda, K., Kuzuya, T., Itakura, J., Nakanishi, H., Takahashi, Y., Kurosaki, M., Asahina, Y., Enomoto, N., Sakamoto, M. and Izumi, N.

Oncology 80 ( 3-4 ) 278 - 288 2011

ISSN  0030-2414

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Objective: Keratin (K) 19 positivity has been reported to be a useful predictive marker for recurrence in patients with hepatocellular carcinoma (HCC) who have undergone hepatic resection. We investigated the clinical usefulness of K19 positivity in patients who had received curative radiofrequency ablation (RFA). Methods: We retrospectively evaluated the clinicopathological features, including imaging and K19 expression, in 246 patients with HCC who were within the Milan criteria and had received curative RFA. Using a two-step insertion method, tumor biopsies were obtained just prior to RFA and were evaluated histologically. Results: Tumor seeding due to liver biopsy and RFA was not observed. Ten patients (4.1%) had K19-positive HCC. Imaging findings were similar between K19-positive and -negative HCC (p = 0.187). Nine out of 10 patients (90%) who had K19-positive HCC had recurrence of HCC after RFA, and intrahepatic recurrences were observed within 12 months in 6 out of 10 (60.0%). K19 positivity was a significant risk factor for recurrence (p < 0.0001) and early recurrence (<1 year after RFA; p = 0.012). K19 expression (p = 0.016) was an independent risk factor for tumor status exceeding the Milan criteria after RFA. Conclusion: Expression of K19 is related to high recurrence of HCC after curative RFA. Copyright (C) 2011 S. Karger AG, Basel

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Reduced transforming growth factor-beta receptor II expression in hepatocellular carcinoma correlates with intrahepatic metastasis

Mamiya, T., Yamazaki, K., Masugi, Y., Mori, T., Effendi, K., Du, W. L., Hibi, T., Tanabe, M., Ueda, M., Takayama, T. and Sakamoto, M.

Laboratory Investigation 90 ( 9 ) 1339 - 1345 2010.09

ISSN  0023-6837

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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<0.001), poor differentiation (P<0.001), portal vein invasion (P = 0.002), intrahepatic metastasis (IM) (P<0.001), and shorter recurrence-free survival (P 0.022). In conclusion, reduced TGFBR2 expression was associated with aggressive features of HCC such as IM, and may represent an immunohistochemical biomarker to detect aggressive HCC. Laboratory Investigation (2010) 90, 1339-1345; doi:10.1038/labinvest.2010.105; published online 7 June 2010

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Molecular diagnosis of multistage hepatocarcinogenesis

Sakamoto, M., Effendi, K. and Masugi, Y.

Jpn J Clin Oncol 40 ( 9 ) 891 - 6 2010.09

ISSN  1465-3621

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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.

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Bmi-1 gene is upregulated in early-stage hepatocellular carcinoma and correlates with ATP-binding cassette transporter B1 expression

Effendi, K., Mori, T., Komuta, M., Masugi, Y., Du, W. L. and Sakamoto, M.

Cancer Science 101 ( 3 ) 666 - 672 2010.03

ISSN  1347-9032

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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. (Cancer Sci 2010; 101: 666-672)

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Production of cells with targeted integration of gene variants of human ABC transporter for stable and regulated expression using the Flp recombinase system

K. Wakabayashi-Nakao, A. Tamura, S. Koshiba, Y. Toyoda, H. Nakagawa and T. Ishikawa

Methods Mol Biol 648   139 - 59 2010

ISSN  1940-6029

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The vector-mediated introduction of cDNA into mammalian cells by calcium phosphate co-precipitation or permeation with lipofectamine is widely used for the integration of cDNA into genomic DNA. Such integration, however, of cDNA occurs randomly at unpredictable sites in the host's chromosomal DNA, and the number of integrated recombinant DNAs is not controllable. To overcome this problem, we developed the Flp-In method to integrate one single copy of cDNA encoding the human ABC transporter ABCG2 into FRT-tagged genomic DNA. Examination of more than 20 metaphase spreads for both fluorescence in situ hybridization (FISH) mapping and multicolor-FISH analysis revealed that ABCG2 cDNA was incorporated into the telomeric region of the short arm on one of chromosomes 12 in Flp-In-293 cells. By using those cells, we investigated the effect of genetic polymorphisms and post-translational modifications of human ABC transporter ABCG2 on the protein expression and degradation. On the basis of our experience, it has been concluded that the Flp recombinase system provides a useful tool to quantitatively analyze the protein stability and endoplasmic reticulum (ER)-associated degradation of proteins like the ABC transporter. This system is also applicable for similar studies of the biogenesis of other proteins using the secretory pathway as well as proteins with other cellular localizations.

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Specific expression of hepatocyte nuclear factor-1βin the ovarian clear cell adenocarcinoma and its application to cytogical diagnosis;

Higashiguchi A,Yamada T,Susumu N,Mori T,Suzuki A,Aoki D,Sakamoto M:

Cancer science. 98 ( 3 ) 387-391 2007.03

Research paper (scientific journal), Single Work

Efficient immortalization of primary human cells by p16INK4a-specific short hairpin RNA or Bmi-1,combined with introduction of hTERT;

Haga K,Ohno SYugawa T,Narisawa-S M,Fujita M,Sakamoto M,Gallaway D,Kiyono T:

Cancer science. 98 ( 2 ) 147-154 2007.02

Research paper (scientific journal), Single Work

cancer stem cells「細胞の不死化・腫瘍化と多分化能の維持」

SAKAMOTO MICHIIE

病理と臨床 25 ( 4 )  2007

Research paper (scientific journal), Joint Work

Gene therapy of liver tumors with liver-specific nanoparticles;

Iwasaki Y,Ueda M,Yamada T,Kondo A,Seno M,Tanizawa K,Kuroda S,Sakamoto M,Kitajima M:

Cancer Gene Therapy 14   74-81 2007

Research paper (scientific journal), Joint Work

Genome-wide array-based comparative genomic hybridization anlysis of pancreatic adenocarcinoma:Identification of genetic indicators that predict patient outcome:

P.loukopoulos,Shiabata T,Katoh H,Kokubu A,Sakamoto M,Yamazaki K,Kosuge T,kanai Y,Hosoda F,Imoto I,Ohki M,Inazawa j,Hirohashi S;

Cancer science 98 ( 3 ) 392-400 2007

Research paper (scientific journal), Single Work

Granulomatous arteritis in cutaneous lesions of churg-strauss syndrome;

Chen K,Sakamoto M,Ikemoto K,Abe R,Shimizu H;

J cutaneous pathology. 134 ( 4 ) 330-337 2007

Research paper (scientific journal), Single Work

「6　肝」第2部　各臓器、疾患で用いられる抗体とその応用

SAKAMOTO MICHIIE

　病理と臨床「臨時増刊号」 25 2007

Research paper (scientific journal), Single Work

Overexpression of cyclase-associated protein 2 in multistage hepatocarcinogenesis

Shibata, R., Mori, T., Du, W. L., Chuma, M., Gotoh, M., Shimazu, M., Ueda, M., Hirohashi, S. and Sakamoto, M.

Clinical Cancer Research 12 ( 18 ) 5363 - 5368 2006.09

ISSN  1078-0432

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Purpose: Hepatocellular carcinoma (HCC) associated with chronic liver disease is known to show an obvious multistage process of tumor progression. We previously identified heat shock protein 70 as a molecular marker of early HCC during investigation of expression profiling in multistage hepatocarcinogenesis. In this report, we examined cyclase-associated protein 2 (CAP2), which is also listed as an up-regulated gene in early HCC.

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Overexpression of Cyclase-Associated Protein 2 in Multistage Hepatocarcinogenesis

Rie Shibata ,Taisuke Mori,Wenlin Du,Makoto Chuma,Masahiro Gotoh,Motohide Shimazu,Masakazu Ueda,Setsuo Hirohashi,SAKAMOTO MICHIIE

Clinical Cancer Research 12 ( 18 ) 5363-5368 2006.09

Research paper (scientific journal), Joint Work

CT Evaluation of the progression of hypoattenuating nodular lesions in virus-related chronic liver disease

Takayasu, K., Muramatsu, Y., Mizuguchi, Y., Okusaka, T., Shimada, K., Takayama, T. and Sakamoto, M.

AJR Am J Roentgenol 187 ( 2 ) 454 - 63 2006.08

ISSN  1546-3141

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OBJECTIVE: The purpose of this study was to clarify the natural outcomes of hypoattenuating nodular lesions in patients with virus-related chronic liver disease depicted on dynamic CT. MATERIALS AND METHODS: Sixty lesions (mean size, 1.3 cm) exhibiting hypoattenuation or isoattenuation in the arterial and delayed phases of dynamic CT were retrospectively evaluated with additional CT (mean, six examinations) for a mean period of 838 days. The primary end point was emergence of hyperattenuating areas within hypoattenuating lesions, a phenomenon called attenuation conversion. Cumulative attenuation conversion rates suggesting rates of malignant transformation were calculated with the Kaplan-Meier method, and factors affecting attenuation conversion rate were analyzed with the Cox proportional hazard model. RESULTS: Thirty-six (60%) of 60 hypoattenuating lesions developed to hyperattenuating lesions, 21 were unchanged, and three disappeared spontaneously. The 36 lesions that became hyperattenuating were divided into two subgroups according to lesion enhancement pattern: hyper-in-hypoattenuating (n = 25) and entirely hyperattenuating (n = 11). The cumulative attenuation conversion rates for the 60 hypoattenuating lesions were 15.8%, 44.3%, and 58.7% at 1, 2, and 3 years. The hyper-in-hypoattenuating lesions showed more rapid progression to entirely enhanced lesions. Positive results for hepatitis C viral antibody (p = 0.028) and initial lesion size (p = 0.007) showed a positive correlation with attenuation conversion rate. CONCLUSION: Hypoattenuating hepatic nodular lesions in chronic liver disease depicted on dynamic CT have high malignant potential and should be followed with special attention to conversion from hypoattenuation to hyperattenuation to determine the optimal timing of treatment.

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Establishment of perineural invasion models and analysis of gene expression revealed an invariant chain (CD74) as a possible molecule involved in perineural invasion in pancreatic cancer.

Koide N, Yamada T, Shibata R, Mori T, Fukuma M, Yamazaki K, Aiura K, Shimazu M, Hirohashi S, Nimura Y, Sakamoto M

Clin Cancer Res. 12 ( 8 ) 2419-26. 2006.05

Research paper (scientific journal), Joint Work

Establishment of perineural invasion models anda analysis of gene ezpression revealed an invariant chain(CD74)as a possible molecule involved in perineural invasion in pancreatic cancer:

Koide N,Yamada T,Shibata R,Mori T,Fukuma M,Yamazaki K,Aiura K,Shimazu M,Hirohashi S,Nimura Y,Sakamoto M;

clin cancer res. 12 ( 8 )  2006

Research paper (scientific journal), Joint Work

A solid pseudopapilary tumor arising from the greater omentum followed by multiple metastases with increasing malignant potential

Hibi T,Ojima H,Sakamoto Y,Kosuge T,Shimada K,Sano T,Sakamoto M,Kitajima M,Yamasaki S:

J Gastroenterol 41   276-181 2006

Research paper (scientific journal), Joint Work

「膵癌の病理学的特性—分子機構の解明—」

SAKAMOTO MICHIIE

画像診断 26 ( 1 )  2006

Research paper (scientific journal), Joint Work

坂元　亨宇　「早期肝細胞癌の病理診断—臨床へのメッセージ」

SAKAMOTO MICHIIE

消化器画像　医学書院 8 ( 1 )  2006

Research paper (scientific journal), Single Work

「2.肝癌の前癌病変・境界病変」

SAKAMOTO MICHIIE

臨床消化器内科 21 ( 7 )  2006

Research paper (scientific journal), Single Work

「胆管浸潤型管内胆管癌の臨床病理学的特徴」

SAKAMOTO MICHIIE

管内胆管癌　消化器画像　医学書院 7 ( 6 )  2005.11

Research paper (scientific journal), Joint Work

Multistep and multicentric development of hepatocellular carcinoma: histological analysis of 980 resected nodules

Oikawa, T., Ojima, H., Yamasaki, S., Takayama, T., Hirohashi, S. and Sakamoto, M.

Journal of Hepatology 42 ( 2 ) 225 - 229 2005.02

ISSN  0168-8278

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Background/Aims: Histological observations support the concept of multistep and multicentric development of hepatocellular carcinoma (HCC) in cases of chronic liver disease. However, the relationship between the incidence of such a modality of development of HCC and the type of background liver disease has not been fully investigated.

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Akt phosphorylation is a risk factor for early disease recurrence and poor prognosis in hepatocellular carcinoma

Nakanishi, K., Sakamoto, M., Yamasaki, S., Todo, S. and Hirohashi, S.

Cancer 103 ( 2 ) 307 - 12 2005.01

ISSN  0008-543X

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BACKGROUND: Patients with hepatocellular carcinoma (HCC) who showed early massive disease recurrence due to hematogenous intrahepatic metastasis after curative resection had a poor prognosis. The authors previously reported that Akt phosphorylation was correlated with hematogenous intrahepatic metastasis, using HCC cell lines. METHODS: The authors analyzed clinicopathologic features and the status of selected biologic markers, including phosphorylated Akt, to identify risk factors for early disease recurrence and poor prognosis in HCC. In the current series, 49 postoperative patients developed intrahepatic disease recurrence within 6 months (Group 1) and 86 patients remained disease recurrence free > 3 years after resection (Group 2). Group 1 was further divided into 2 subgroups: 19 patients who died of disease recurrence within a year after resection (Group 1A) and 27 patients who survived > 1 year (Group 1B). RESULTS: Using univariate analysis, the risk factors for early disease recurrence were tumor size, macroscopic classification, tumor differentiation, microscopic capsule infiltration, microscopic portal vein (MPV) invasion, microscopic intrahepatic metastasis (MIM), and positive immunostaining for phosphorylated Akt, Ki-67, and p53 (P < 0.05). The risk factors for poor prognosis were the number of intrahepatic metastases, tumor differentiation, and positive immunostaining for phosphorylated Akt and Ki-67 (P < 0.05). Multivariate analysis revealed that the risk factors for early disease recurrence were MPV invasion, MIM, and positive immunostaining for phosphorylated Akt, and that the risk factors for poor prognosis were positive immunostaining for phosphorylated Akt and Ki-67 (P < 0.05). CONCLUSIONS: The current clinical study showed the critical involvement of Akt phosphorylation in the aggressiveness of HCC. The potential benefits of surgery should be assessed carefully in patients with any of these risk factors.

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Akt Phosphorylation is risk factor for early disease recurrence and poor prognosis in hepatocellular caricinoma ;

Nakanishi K,Sakamoto M,Yamasaki S,Todo S,Hirohashi S:

cancer 103 ( 2 )  2005.01

Research paper (scientific journal), Joint Work

Long-term survival of intrahepatic cholangiocarcinoma with hilar lymph node metastasis anda portal vein invovment:

Akatsu T,Shimazu M,Kawachi S,Tanabe M,Aiura K,Wakabayashi S,Ueda M,Sakamoto M,Kitajima M:

hepato-gastroenterology 52   606-609 2005

Research paper (scientific journal), Joint Work

Clinical and pathologic signigicance of activation of signal transducer and activator of transcription 3 in prostate cancer:

Horinaga M,Okita H,Nakashima J,Kanao K,Sakamoto M,Murai M:

urology. 66 ( 3 )  2005

Research paper (scientific journal), Joint Work

Domain-specific function of ShcC docking protein in neuroblastoma cells:

Miyake I,Hakomori Y,Misu Y,Nakadate H,Matsuura N,Sakamoto M,Sakai R:

oncogene. 24   3206-3215. 2005

Research paper (scientific journal), Joint Work

Fukusato T,Fukushima J,Shiga J,Takahashi Y,Nakano Interobserver variation in the hitopathological assessment of nonalcoholic steatohepatitis;

T,Maeyama S,Masayuki U,Ohbu M,Matsumoto T,Matsumoto K,Hano H,Sakamoto M,Kondo F,Komatsu S,Ishikawa T,Ohtake H,Takikawa H,Yoshimura K,Liver disease working group-kanto;

Hepatology Research 33   122-127, 2005

Research paper (scientific journal), Joint Work

Genetic profile of hepatocellular carcinoma revealed by array-based comparative genomic hybridization:Identification of genetic indicators to predict patient outcome:

Katoh H,Shibata T,Kokubu A,Ojima H,P.Loukopoulos,Kanai Y,Kosuge T,Fukayama M,Kondo T,Sakamoto m,Hosoda F,Ohiki M,Imoto I,Inazawa J,Hirohashi S;

Journal hepatology. 43   863-874. 2005

Research paper (scientific journal), Joint Work

肝腫瘍の術中迅速診断　肝生検診断Ⅰ−腫瘍性疾患−

SAKAMOTO MICHIIE

病理と臨床・別刷 23 ( 2 )  2005

Research paper (scientific journal), Single Work

Primary bone carcinosarcoma:Chondrosarcoma and squamous cell carcinoma with keratin pearl formation:

Shiraishi J,Mukai M,Yabe H,Shibata R,Yamada T,Miura K,Anazawa Morioka H,Sakamto M:

Pathology International 55   504-409 2005

Research paper (scientific journal), Joint Work

Multistep and multicentric development of hepatocellular carcinoma:histological analysis of 980 resected nodules .

Oikawa T,Ojima H,Yamazaki S,Takayama T,Hirohashi S,Sakamoto M:

Journal of Hepatology 42   225-229 2005

Research paper (scientific journal), Joint Work

Extramedullary myeloid tumor (EMMT)of the gallbladder .

Ojima H,Hasegawa T,Matsuo Y,Sakamoto M:

J Clin Pathol . 58   211-213 2005

Research paper (scientific journal), Joint Work

Spontaneous rupture of a mass-forming type peripheral cholangiocarcinoma:

Akatsu T,Ueda M,Shimazu M,Kawauchi S,Tanabe M,Aiura K,Wakabayashi G,Sakamoto M,Matsuo R:

Journal of Gastroenterology and Hepatology. 20   162-168 2005

Research paper (scientific journal), Joint Work

Combination of hTERT and bimi-1,E6,or E7,induces prolongation of the life span of bone marrow stromal cells from an elderly donor without affecting their neurogenic protential:

Mori T,Kiyono T,Imabayashi H,Takeda Y,Tsuchiya K,Miyoshi S,Makino H,Matsumoto K,Saito H,Ogawa S,Sakamoto M,Hata J,Umezawa A:

molecular and cellular biology. 25 ( 12 ) 5182-5195 2005

Research paper (scientific journal), Joint Work

Extramedullary myeloid tumor (EMMT)of the gallbladder .

Ojima H,Hasegawa T,Matsuo Y,Sakamoto M:

J Clin Pathol . 58   211-213 2005

Research paper (scientific journal), Joint Work

Proposal of histological criteria for intraepithelial atypical/proliferative biliary epithelial lesions of the bile duct in hepatolithiasis with respect to cholangiocarcinoma:Preliminary report based on interobserver agreement

Yoh Z,Aishima S,Ajioka Y,Haratake J,Kage M,Kondo F,Nimura Y,Sakamoto M,Sasaki M,Shimamatsu K,Wakasa K,Young N.P,Ming F.C,Atomi Y,Nakanuma Y:

Pathology international. 55   180-188, 2005

Research paper (scientific journal), Joint Work

Overexpression of cortactin is involved in motility and metastasis of hepatocellular carcinoma

Chuma, M., Sakamoto, M., Yasuda, J., Fujii, G., Nakanishi, K., Tsuchiya, A., Ohta, T., Asaka, M. and Hirohashi, S.

Journal of Hepatology 41 ( 4 ) 629 - 636 2004.10

ISSN  0168-8278

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Background/Aims: The molecular basis of the metastasis of hepatocellular carcinoma (HCC) is not fully understood. The aim of this study was to elucidate the crucial genes involved in metastasis of HCC.

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Orthotopic Transplantation Models of Pancreatic Adenocaricinoma Derived From Cell Lines and Primary Tumors and displaying barying metastatic activity;

Panayiotis L DVN,Kanetaka K,Takamura M,Shibata T,Sakamoto M,Hirohashi S:

pancreatic cancer orthotopic models 29 ( 3 ) 2004 2004.10

Research paper (scientific journal), Joint Work

Expression profiling in hepatocellular carcinoma with intrahepatic metastasis: identification of high-mobility group I(Y) protein as a molecular marker of hepatocellular carcinoma metastasis

Chuma, M., Saeki, N., Yamamoto, Y., Ohta, T., Asaka, M., Hirohashi, S. and Sakamoto, M.

Keio J Med 53 ( 2 ) 90 - 7 2004.06

ISSN  0022-9717

　View Summary

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Its high mortality rate is mainly a result of intra-hepatic metastases. To investigate the detailed genetic mechanisms in cancer metastasis, we compared the expression profiles of 20 HCCs with intrahepatic metastasis and 10 HCCs without intrahepatic metastasis using an oligonucleotide array. Of the approximately 12,600 genes that were analyzed, we identified 34 genes whose expression levels were significantly correlated with intrahepatic metastasis (P<0:05). Of these genes, we further investigated the expression of high-mobility group I(Y) [HMG-I(Y)] protein. Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) confirmed that HMG-I(Y) was upregulated in HCC with intrahepatic metastasis, compared to its level in HCC without intrahepatic metastasis. Further immunohistochemical examination of HMG-I(Y) revealed a significant overexpression in HCC with intrahepatic metastasis, compared with that in HCC without intrahepatic metastasis (P<0:05). These results indicate that the molecular signatures of HCC with intrahepatic metastasis and of HCC without intrahepatic metastasis are clearly different. HMG-I(Y) expression was associated with intrahepatic metastasis and may be a predictive marker of HCC intrahepatic metastasis.

Can the life span of human marrow stromal cells be prolonged by bmi-1 E6,E7and/or telomerase without affecting cardiomyogenic defferentiation ;

Takeda Y,Mori T,Imabayashi H,Kiyono T,Gojo S,Miyoshi S,Hida N,Ita M,Segawa S,Ogawa S,Sakamoto M,Nkamura S,Umezawa A:

J.Gene Med. 0   833-845 2004.06

Research paper (scientific journal), Single Work

肝門部胆管癌進展様式の病理学的特徴：肝門部胆管癌の進展度診断−画像でどこまでわかるか：

SAKAMOTO MICHIIE

医学書院  2004.05

Research paper (scientific journal), Joint Work

Clinical significance of dysadherin expression in gastric cancer patients;

'Shimada Y, Yamasaki S, Hashimoto Y, Ito T, Kawamura J, Soma T, Ino Y, Nakamnishi Y, Sakamoto Michiie, Hirohashi Setsuo, Imamura M'

clinical cancer research 2818 ( 10 ) 2818-2823 2004.04

Research paper (scientific journal), Single Work

肝細胞癌の多段階発癌とフェノタイプ、

SAKAMOTO MICHIIE

肝胆膵 48 ( 4 ) 2004 2004.04

Research paper (scientific journal), Joint Work

肝細胞癌の病理　肝細胞性結節性病変の鑑別を中心に　：

SAKAMOTO MICHIIE

臨床放射線 49 ( 11 )  2004

Research paper (scientific journal), Joint Work

Pedunculated　 angiomyolipoma of the liver with a predominant pelioid pattern

'Akatsu Tomotaka, Sakamoto Michiie, Shimazu M, Kitajima Masaki'

Virchows Archiv    444;467-469 2004

Research paper (scientific journal), Single Work

Tumor angiogenesis in the bone marrow of multiple myeloma patients and its alteration by thalidomide treatment

'Wenlin Du, Hattori Y, Hashiguchi Akinorik, Kondoh Keisuke, Hozumi Nobumichi, Ikeda Y, Sakamoto Michiie, Hata Junichi, Yamada Taketo'

pathology International  ( 54 ) 285-294 2004

Research paper (scientific journal), Single Work

Cusom-shaping system gor bon eregeneration by seeding marrow stromal cells onto a web-like biodegradable hybrid sheet

'Tsuchiya Kouhei, Mori Taisuke, Chen G, Ushida T, Tateishi T, Matsuo T, Sakamoto Michiie, Umezawa Akihiro'

Cell Tissue Research  ( 316 ) 141-153 2004

Research paper (scientific journal), Single Work

5-Aza-2’-deoxycytidine restores the E-cadherin system in E-cadherin-silenced cancer cells and reduces cancer metastasis;

'J-S-Nam, Ino Y, kanai Yae, Sakamoto Michiie, Hirohashi Setsuo'

Clnical&Experimental Metastasis 21   29-56 2004

Research paper (scientific journal), Single Work

Expression profiling in hepatocellular carcinoma with intrahepatic metastasis identification of high-mobility groupⅠ(Y)protein as a molecular marker of hepatocellular carcinoma metastasis

Chuma M,Saeki N,Yamamoto Y,Ohta T,Asaka M,Hirohashi S,Sakamoto M:

Keio Journal of Medicine, l53 ( 2 ) 90-97 2004

Research paper (scientific journal), Joint Work

Can the life span of human marrow stromal cells be prolonged by bmi-1 E6,E7and/or telomerase without affecting cardiomyogenic defferentiation ;J

Takeda Y,Mori T,Imabayashi H,Kiyono T,Gojo S,Miyoshi S,Hida N,Ita M,Segawa S,Ogawa S,Sakamoto M,Nkamura S,Umezawa A:

Gene Med. 6   833-845 2004

Research paper (scientific journal), Joint Work

Lamin 5β3 and γ2 chains are frequently coexpressed in cancer cells;

Akimoto S,Nakanishi Y,Sakamoto M,Kanai Y,Hirohashi S:

Pathology International 54   688-692 2004

Research paper (scientific journal), Joint Work

Dysadherin expression facilitates cell motility and metastatic potential human pancreatic cancer cells;

Shimamura T,Yasuda J,Ino M,Tsuchiya A,Najima A,Sakamoto M,Kanai Y,Hirohashi S:

cancer research 64   6989-6995, 2004

Research paper (scientific journal), Joint Work

Prognostic significance of deysadherin expression in esophageal squamous cell carcinoma;

Shimada Y,Hashimoto Y,Kan T,Kawamura J,Okumura T,Soma T,Kondo K,TerataniN,Watanabe G,Ino Y,Sakamoto M,Hirohashi S,Imamura M:

oncology 67   73-80 2004

Research paper (scientific journal), Joint Work

High-grade dysplasia associated with fundic gland polyposis in a familial adenomatous polyposis patient,with special reference to APC mutation profiles.

Sekine S, Shimoda T, Nimura S, Nakanishi Y, Akasu T, Katai H, Gotoda T, Sakamoto M, Hirohashi S,

Modern Pathology, 17   1421-1426 2004

Research paper (scientific journal), Joint Work

Overexpression of cortactin is involved in motility and metastasis of hepatocellular carcinoma.

Chuma M, Sakamoto M, Yasuda J, Fujii G, Nakanishi K, Tsuchiya A, Ohta T, Asaka M, Hirohashi S,

Journal of Hepatology. 41   626-636 2004

Research paper (scientific journal), Joint Work

Reduced expression of liver-intestine cadherin is associated with progression and lymph node metastasis of human colorectal carcinoma.

Takamura M,Ichida T,Matsuda Y,Kobayashi M,Yamagiwa S,Genda T,Shioji K,Hashimoto S,Nomoto M,Hatakeyama K,Ajioka Y,Sakamoto M,Hirohashi S,Aoyagi Y:

Cancer Letters . 212   253-259 2004

Research paper (scientific journal), Joint Work

Squamous cell carcinoma of the liver originating from non-parasitic cysts after a 15 year follow-up:

Yagi H,Ueda M,Kawachi S,Tanabe M,Aiura K,Wakabayashi G,Shimazu M,Sakamoto M,kitajima M:

European journal of gastroentelrology &hepatology 16 ( 10 )  2004

Research paper (scientific journal), Joint Work

Squamous cell carcinoma of the liver originating from non-parasitic cysts after a 15 year follow-up:

Yagi H,Ueda M,Kawachi S,Tanabe M,Aiura K,Wakabayashi G,Shimazu M,Sakamoto M,kitajima M:

European journal of gastroentelrology &hepatology 16 ( 10 )  2004

Research paper (scientific journal), Joint Work

prognostic signficane of dysadherin expression in Tongue cancer :

Nanishi Y,Akimoto S,Sato Y,Kanai Y,Sakamoto M,Hirohashi S:

Immunohistochemical analysis of 91 casesappl immunohistochem mol morphol 12 ( 4 )  2004

Research paper (scientific journal), Joint Work

病理からのフィードバック　肝細胞癌の肉眼型と大腸癌肝細胞転移の進展様式、

SAKAMOTO MICHIIE

医学書院、 5 ( 3 )  2004

Research paper (scientific journal), Joint Work

Expression profiling in multistage hepatocarcinogenesis: Identification of HSP70 as a molecular marker of early hepatocellular carcinoma.

Chuma, M., Sakamoto, M., Hige, S. H., Asaka, M. and Hirohashi, S.

Hepatology 38 ( 4 ) 402a - 402a 2003.10

ISSN  0270-9139

Proteomic Analysis of Intestinal Epithelial Cells expressing stabilized β-Catenin1;

'Seike M, Kondo T, Mori Y, Gemma A, Kondoh S, Sakamoto M, Yamada Taketo, Hirohashi Setsuo'

Cancer Research 63   4641-4647 2003.08

Research paper (scientific journal), Single Work

Late Complication of a large simple cyst of the liver mimicking cystadenocarcinoma after sclerotherapy;

'Takayasu K, Mizuguchi Y, Yamazaki S, Sugiura T, Sato Chise, Sakamoto Michiie'

AJR 181 2003.08

Research paper (scientific journal), Single Work

Alpha-Fetoprotein-Producing Caricinoma of the Colon:

'Yachida S, Fukushima N, Nakanishi Y, Akatsu T, Kitamura H, Sakamoto Michiie, Shimoda'

Dis Colon Rectum 46 ( 6 )  2003.06

Research paper (scientific journal), Single Work

'EBP50,a β-Catenin-Associating Protein,Enhances Wnt Signaling and Is Over-expresseed in Hepatocellular Carcinoma'

'Shibata Rie, Chuma M, Kokubu A, Sakamoto Michiie, Hirohashi Setsuo'

Hepatology 0 ( 0 )  2003.06

Research paper (scientific journal), Single Work

Overexpression of orphan G-Protein-coupled receptor, Gpr49, in human hepatocellular carcinomas with beta-catenin mutations

Yamamoto, Y., Sakamoto, M., Fujii, G., Tsuiji, H., Kanetaka, K., Asaka, M. and Hirohashi, S.

Hepatology 37 ( 3 ) 528 - 533 2003.03

ISSN  0270-9139

　View Summary

To identify the genes responsible for carcinogenesis and progression of hepatocellular carcinoma (HCC), we screened differentially expressed genes in several human HCC cell lines. Among these genes, Gpr49 was up-regulated in PLC/PRF/5 and HepG2. Gpr49 is a member of the glycoprotein hormone receptor subfamily, which includes the thyroid-stimulating hormone receptor (TSHR). However, Gpr49 remains to be an orphan G-protein-coupled receptor. By real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis, overexpression (>3-fold increase compared with the corresponding noncancerous liver tissue) of Gpr49 mRNA was observed in 18 of 38 (47%) HCCs compared with corresponding noncancerous livers. Clinicopathologically, overexpression of Gpr49 was frequently observed in HCC with mutation in beta-catenin exon 3 (14 of 16 cases, 87.5%). Moreover, introduction of mutant beta-catenin into mouse hepatocytes in culture caused up-regulation of the Gpr49 mouse homologue. Therefore, Gpr49 is likely to be a target gene activated by Wnt-signaling in HCC. In conclusion, although much is still unknown, Gpr49 may be critically involved in the development of HCCs with beta-catenin mutations and has the potential to be a new therapeutic target in the treatment of HCC.

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Prognostic singnificance of dysadherin expression in advanced colorectal carcinoma

Aoki S, Shimamura T, Shibata T, Nakanishi Y, Moriya Y, Kitajima M, Sakamoto M, Hirohashi S

British journal of cancer 88 ( 5 ) 726-732 2003.03

Research paper (scientific journal), Joint Work

性幹細胞（組織幹細胞）の分離，増殖，分化誘導体

Hashiguchi Akinori, Sakamoto Michiie, Umezawa Akihiro

日本臨牀 61 ( 3 ) 390-395 2003.03

Research paper (scientific journal), Joint Work

Dysadherin Overepression in Pancreatic Ductal Adenocarcinoma Reflects Tumor Aggressiveness:Relationship to E-Cadherin Expression

Shimamura Takeshi, Sakamoto Michiie, Ino Yoshinori, Sato Yasuto, Shimada Kazuaki, Kasuge Tohru, Sekihara Hisahiko, Hirohashi Setsuo

Journal of clinical oncology 21 ( 4 ) 659-667 2003.02

Research paper (scientific journal), Joint Work

A faithful method for PCR-mediated global mRNA amplification and itsintegration into microarray analysis on laster-captured cells

Aoyagi Kazuhiko, Tatsuta Tatsuta, Nishigaki Michiko, Akimoto Chikako, Tanabe Chikako, Omoto Yoko, Hayashi Shin-ichi, Sakamoto Hiromi, Sakamoto Michiie, Yoshida Teruhiko, Terada Masaaki, Sasaki Hiroki

Biochemical and biophysical research communications 300 ( 4 ) 915-920 2003.01

Research paper (scientific journal), Joint Work

Overexpression of orphan G-Protein-Coupled Receptor,Gpr49,in human hepatocellular carcinoma with β-catenin mutations

Yamamoto Y, Sakamoto Michiie, Fujii Gen, Tsuiji Hitomi, Kanetaka Kenzo, Asaka Masahiro, Hirohashi Setsuo

Hepatology 37 ( 3 ) 528-533 2003

Research paper (scientific journal), Joint Work

Expression profiling in ovarian clear cell carinoma identification of Hepatocyte nuclear factor-1 β as a Molecular marker and a possible molecular target for therapy of ovarian clear cell carcinoma;

'Tsuchiya A, Sakamoto Michiie, Yasuda J, chuma Makoto, Ohta T, Ohki M, Yasugi T, Taketani Y, Hirohashi Setsuo'

American journal of pathology 163 ( 6 )  2003

Research paper (scientific journal), Single Work

Mutation of the DNA metyltransferase(DNMT)1 gene in human colorectal cancers;

'Kanai Yae, Ushijima S, Nakanishi Y, Sakamoto Michiie, Hirohashi Setsuo'

Cancer letters 192   75-82 2003

Research paper (scientific journal), Single Work

Primary carcinoma tumor in a polycystic kidney;

'Shibata Rie, Okita Hajime, Shimoda Masayuki, Asakura H, Murai M, Sakamoto Michiie, Hata Junichi'

Pathology international 53   317-322 2003

Research paper (scientific journal), Single Work

A case of scleosing hemangioma of the lung presenting as gigantic tumor occupying the left thoracic cavity;

'Shibata Rie, Mukai M, Okada Yasunori, Sakamoto Michiie, Yamauchi T, Kobayashi K'

Virchows Arch 442   409-411 2003

Research paper (scientific journal), Single Work

Expression profiling in ovarian clear cell carinoma identification of Hepatocyte nuclear factor-1 β as a Molecular marker and a possible molecular target for therapy of ovarian clear cell carcinoma;

'Tsuchiya A, Sakamoto Michiie, Yasuda J, Chuma Makoto, Ohta T, Ohki M, Yasugi T, Taketani Y, Hirohashi Setsuo'

American journal of pathology 163 ( 6 )  2003

Research paper (scientific journal), Single Work

Prediction of Invasive Activities in Hepatocellular Carcinomas with Special Reference to α-Fetoprotein and Des-γ-carboxyprothrombin

'Gotoh M, Nakatani T, Masuda T, Mizuguchi Y, Sakamoto Michiie, Tsuchiya R, Kato H, Furuta K'

Japanese Journal of Clinical Oncology 33 ( 10 ) 522-526 2003

Research paper (scientific journal), Single Work

Possible involvement of tetraspanin CO-029 in hematogenous intrahepatic metastasis of liver cancer cells;

'Kanetaka K, Sakamoto Michiie, Yamamoto Y, Takamura M, Kanematsu T, Hirohashi Setsuo'

Journal of Gastroenterology and Hepathology 18   1309-1314 2003

Research paper (scientific journal), Single Work

'Expression of Different Glycoforms of Membrane Mucin (MUC1)and Secretory Mucin(MUC2,MUC5AC and MUC6)in Pancreatic Neoplasms'

'Horinouchi M, Nagata K, Nakamura A, Goto M, Takao S, Sakamoto Michiie, Fukushima N, Miwa A, Irimura T, Imai K, Sato, Yonezaki S'

Acta Histochemica et Cytochemica 36 ( 5 ) 443-453 2003

Research paper (scientific journal), Single Work

Evaluation of a Whole-Genome Amplification Method Based on Adaptor-Ligation PCR of Randomly Sheared Genomic DNA

'Tanabe C, Aoyagi K, Sakiyama T, Kohno T, Yanagitani N, Akimoto S, Sakamoto Michiie, Sakamoto H, Jun Yokota, Ohki M, Terada M, Yoshida T'

Genes chromosomed&cancer 38   168-176 2003

Research paper (scientific journal), Single Work

Nemo &#8211;like kinase induces apoptosis in DLD-1 human colon cancer cells

'Yasuda J, Tsuchiya A, Yamada Taketo, Sakamoto Michiie, Sekiya T, Hirohashi Setsuo'

Biochemical and Biophysical Research Communications 308   227-233 2003

Research paper (scientific journal), Single Work

Increaseda protein expression of DNA methyltranseferase(DNMT)1 is significantly correlated with the malignant potential and poor prognosis of human hepatocellular carcinomas;

'Saito Y, Kanai Yae, Nakagawa T, Sakamoto Michiie, Saito H, Ishii H, Hirohashi Setsuo'

International Journal of Cancer 105   527-532 2003

Research paper (scientific journal), Single Work

Expression of liver-intestine cadherin and its possible interaction with galectin-3 in ductal adenocarcinoma of the pancreas;

'Takamura M, Sakamoto Michiie, Ino Y, Shimamura T, Ichida T, Asakura H, Hirohashi Setsuo'

Cancer science 194 ( 5 ) 425-430 2003

Research paper (scientific journal), Single Work

β-Catenin mutations in pulmonary blastomas:association with morule formation;

'Sekine S, Shibata Rie, Matsuo Y, Maeshima A, Ishii G, Sakamoto Michiie, Hirohashi Setsuo'

Journal of Pathology    214-221 2003

Research paper (scientific journal), Single Work

'Aberrantο-glycosylation inhibits stable expression of dysadherin,a carcinoma-associated antigen,and facilitates cell-cell adhesion;'

'Tsuji H, Takasaki S, Sakamoto Michiie, Irimura T, Hirohashi Setsuo'

Glycobiology 13 ( 7 ) 521-527 2003

Research paper (scientific journal), Single Work

Clear Cell Endocrine Tumor of the Pancreas Which is Not Associaated with von Hippel-Lindau Disease:

'Nunobe S, Fukushima N, Yachida S, Shimada K, Kosuge T, Sakamoto Michiie'

Report of Case:Surg Today 33   470-474 2003

Research paper (scientific journal), Single Work

Craniopharyngiomas of Adamantinomatous Type Harbor β-Catenin Gene Mutations

Sekine Shigeki, Shibata Tatsuhiro, Kokubo Akiko, Morishita Yukio, Noguchi Masayuki, Nakanishi Yukihiro, Noguchi Masayuki, Nakanishi Yukihiro, Sakamoto Michiie, Hirohashi Setsuo

American journal of pathology 161 ( 6 ) 1997-2001 2002.12

Research paper (scientific journal), Joint Work

Satellite lesions in patients with small hepatocellular carcinoma with reference to clinicopathologic features

Okusaka, T., Okada, S., Ueno, H., Ikeda, M., Shimada, K., Yamamoto, J., Kosuge, T., Yamasaki, S., Fukushima, N. and Sakamoto, M.

Cancer 95 ( 9 ) 1931 - 7 2002.11

ISSN  0008-543X

　View Summary

BACKGROUND: It is not rare to find satellite lesions in patients with small hepatocellular carcinoma (HCC). The purpose of this study was to elucidate the factors associated with satellite lesions in these patients. METHODS: We investigated the prevalence of satellite lesions, the relationship of clinicopathologic factors to satellite lesions, and the distance from the main tumor to the satellite lesion in 149 patients. Patients, who had a solitary HCC of 3.0 cm or less in diameter but no satellite lesions on preoperative imaging procedures, underwent potentially curative resection. The main tumors were macroscopically classified into four groups: early HCC, a vaguely nodular type showing preservation of the preexisting liver structure; single nodular type; single nodular type with extranodular growth; and confluent multinodular type. RESULTS: Of 149 resected specimens, 28 (19%) showed satellite lesions. Of the clinicopathologic factors investigated, the macroscopic type and tumor differentiation were significantly associated with the prevalence of satellite lesions. Both the single nodular type with extranodular growth and the confluent multinodular type showed satellite lesions more frequently than the early HCC and the single nodular type. A significantly higher prevalence of satellite lesions was observed in poorly differentiated HCC than in well and moderately differentiated HCC. The satellite lesions were located 0.5 cm or less from the main tumor in 8 (33%) specimens, 0.6-1.0 cm in 12 (50%), and 1.1-2.0 cm in 4 (17%). No identifiable factors were significantly related to the distance from the main tumor to the satellite lesion. However, all satellite lesions located more than 1.0 cm from the main tumor coexisted with poorly differentiated HCC, which were the single nodular type with extranodular growth or the confluent multinodular type. CONCLUSION: In the single nodular type with extranodular growth, confluent multinodular type, and poorly differentiated HCC, extensive treatment achieving a large safety margin and/or frequent posttreatment follow-up examinations may be needed because of the high prevalence of satellite lesions.

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Satellite lesions in patientswith small hepatocellular carcinoma with reference to clinicopathologic features

Okusaka Takuji, Okada Shuichi, Ueno Hideki, Ikeda Masafumi Shimada Kazuaki, Yamamoto Junji, Kosuge Tomoo, Yamazaki Susumu, Fukushima Noriyuki, Sakamoto Michiie

Cancer 95 ( 9 ) 1931-1937 2002.11

Research paper (scientific journal), Joint Work

Minute pancreatic adenocarcinoma presenting with stenosis of the main pancreatic duct

Yachida Shinichi, Fukushima Noriyoshi, Nakanishi Kazuaki, Shimada Kazuaki, Kosuge Tomoo, Sakamoto Michiie

Pathology international 52 ( 9 ) 607-611 2002.09

Research paper (scientific journal), Joint Work

β-catenin mutations in sporadic fundic gland polyps

Sekine Shigeki, Shibata Tatsuhiro, Yamauchi Yuko, Nakanishi Yukihiro, Shimoda Tadakazu, Sakamoto Michiie, Hirohashi Setsuo

Virchows Archiv 440 ( 4 ) 381-386 2002.09

Research paper (scientific journal), Joint Work

Less aggressive features of colorectal cancer with liver metastases showing macroscopic intrabiliary extension

Kubo Makoto, Sakamoto Michiie, Fukushima Noriyoshi, Yachida Shinichi, Nakanishi Yukihiro, Shimoda Tadakazu, Yamamoto Junji, Moriya Yoshihiro, Hirohashi S

Pathology international 52 ( 8 ) 514-518 2002.08

Research paper (scientific journal), Joint Work

Clinicopathological Siginificance of Galectin-3 Expression in Ductal Adenocarcinoma of the Pancreas

Shimamura Takeshi, Sakamoto Michiie, Ino Yoshinori, Shimada Kazuaki, Kosuge Tomoo, Sato Yasuto, Tanaka Katsuaki, Sekihira Hisahiko, Hirohashi Setsuo

Clinical cancer research 8 ( 8 ) 2570-2575 2002.08

Research paper (scientific journal), Joint Work

Pancreatic metastasis from renal cell carcinoma extending into the main pancreatic duct:a Case Report

Yachida Shinichi, Fukushima Noriyoshi, Kanai Yae, Nimura Satoshi, Shimada Kazuaki, Yamamoto Junji, Sakamoto Michiie

Japanese journal of clinical oncology 32 ( 8 ) 315-317 2002.08

Research paper (scientific journal), Joint Work

Target disruption of the mutantβ-Catenin gene in colon cancer cell line HCT116:preservation of its malignant phenotype

Sekine Shigeki, Shibata Tatsuhiro, Sakamoto Michiie, Hirohashi Setsuo

Oncogene 21 ( 38 ) 5906-5911 2002.08

Research paper (scientific journal), Joint Work

Overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, associated with DNA hypomethylation on pericentromeric satellite regions during human hepatocarcinogenesis

Saito, Y., Kanai, Y., Sakamoto, M., Saito, H., Ishii, H. and Hirohashi, S.

Proc Natl Acad Sci U S A 99 ( 15 ) 10060 - 5 2002.07

ISSN  0027-8424

　View Summary

DNA hypomethylation on pericentromeric satellite regions is an early and frequent event associated with heterochromatin instability during human hepatocarcinogenesis. A DNA methyltransferase, DNMT3b, is required for methylation on pericentromeric satellite regions during mouse development. To clarify the molecular mechanism underlying DNA hypomethylation on pericentromeric satellite regions during human hepatocarcinogenesis, we examined mutations of the DNMT3b gene and mRNA expression levels of splice variants of DNMT3b in noncancerous liver tissues showing chronic hepatitis and cirrhosis, which are considered to be precancerous conditions, and in hepatocellular carcinomas (HCCs). Mutation of the DNMT3b gene was not found in HCCs. Overexpression of DNMT3b4, a splice variant of DNMT3b lacking conserved methyltransferase motifs IX and X, significantly correlated with DNA hypomethylation on pericentromeric satellite regions in precancerous conditions and HCCs (P = 0.0001). In particular, the ratio of expression of DNMT3b4 to that of DNMT3b3, which is the major splice variant in normal liver tissues and retains conserved methyltransferase motifs I, IV, VI, IX, and X, showed significant correlation with DNA hypomethylation (P = 0.009). Transfection of human epithelial 293 cells with DNMT3b4 cDNA induced DNA demethylation on satellite 2 in pericentromeric heterochromatin DNA. These results suggest that overexpression of DNMT3b4, which may lack DNA methyltransferase activity and compete with DNMT3b3 for targeting to pericentromeric satellite regions, results in DNA hypomethylation on these regions, even in precancerous stages, and plays a critical role in human hepatocarcinogenesis by inducing chromosomal instability.

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Hepatic nodules with early enchancement during computed tomography portography:Report of six cases

Takayasu Kenichi, Muramatsu Yukio, Wakao Fumihiko, Mizuguchi Yasunori, Iwata Ryoko, Maeda Tetsuo, Moriyama Noriyuki, Sakamoto Michiie

Journal of gastroenterology and hepatology 17 ( 7 ) 779-784 2002.07

Research paper (scientific journal), Joint Work

Correlation between laminin-5γ2 chain expression and epidermal growth factor receptor expression and its clinicopathologic significance in squamous cell carcinoma of the tongue

Katoh Kazuhide, Nakanishi Yukihiro, Akimoto Shingo, Yoshimura Kimio, Takagi Minoru, Sakamoto Michiie, Hirohashi Setsuo

Oncology 62 ( 4 ) 318-326 2002.07

Research paper (scientific journal), Joint Work

Overexpression of a splice variant of DNA methyltransferase 3b,DNMT3b4,associated with DNA hypomethylation on pericentromeric satellite regions during human hepatocarcinogenesis

Saito Yoshimasa, Kanai Yae, Sakamoto Michiie, Saito Hidetsugu, Ishii Hiromasa, Hirohashi Setsuo

Proceedings of the National Academy of Science of the United States of America 99 ( 15 ) 10060-10065 2002.07

Research paper (scientific journal), Joint Work

Overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, associated with DNA hypomethylation on pericentromeric satellite regions during human hepatocarcinogenesis

Saito Yoshimasa, Kanai Yae, Sakamoto Michiie, Saito Hidetsugu, Ishii Hiromasa, Hirohashi Setsuo

Proceedings of the National Academy of Sciences of the United States of America 99 ( 15 ) 10060-10065 2002.07

Research paper (scientific journal), Joint Work

Phosphogyceride crystal deposition disease presenting as multiple paragastric masses

Yachida Shinichi, Fukushima Noriyoshi, Nakanishi Yukihiro, Nimura S, Sato M, Shimada K, Miura K, Sakamoto Michiie

Digestive diseases and sciences 47 ( 6 ) 1413-1418 2002.06

Research paper (scientific journal), Joint Work

Mixed mucinous-type and non-mucinous-type adenocarcinoma of the lung: immunohistochemical examination and K-ras gene mutation

Maeshima Aratumi, Sakamoto Michiie, Hirohashi Setsuo

Virchows Archiv 440 ( 6 ) 598-603 2002.06

Research paper (scientific journal), Joint Work

Critical involvement of the phosphatidylinositol 3-kinase/Akt pathway in anchorage-independent growth and hematogeneous intrahepatic metastasis of liver cancer

Nakanishi, K., Sakamoto, M., Yasuda, J., Takamura, M., Fujita, N., Tsuruo, T., Todo, S. and Hirohashi, S.

Cancer Res 62 ( 10 ) 2971 - 5 2002.05

ISSN  0008-5472

　View Summary

In the multistep process of metastasis, "anchorage-independent growth," where cancer cells need to survive without cell-substratum interaction, is supposed to be important. In this study, we found that anchorage-independent growth analyzed using the soft agar colony formation assay correlated with hematogeneous intrahepatic metastasis of liver cancer cell lines and also Akt activation status. Two highly metastatic liver cancer cell lines showed high Akt activity and formed many colonies in soft agar, whereas three nonmetastatic cell lines showed less Akt activity and formed fewer colonies. Inhibition of Akt activation in the highly metastatic cell line Li7 by transfection with kinase-dead Akt or the phosphatidylinositol 3-kinase inhibitor, LY294002, resulted in formation of fewer colonies in soft agar than was the case with control cells. Moreover, in orthotopic implantation model, this inhibition resulted in a reduced rate of hematogeneous intrahepatic metastasis. These findings indicated that anchorage-independent growth regulated by phosphatidylinositol 3-kinase/Akt pathway plays a critical role in metastasis, and that this could be a potential therapeutic target to combat metastasis.

Critical involvement of the phosphatidylinositol 3-kinase/Akt pathway in anchorage-independent growth and hematogeneous intrahepatic metastasis of liver cancer

Nakanishi Kazuaki, Sakamoto Michiie, Yasuda Jun, Takamura Masaaki, Fujita Naoya, Tsuruo Takashi, Todo Satoru, Hirohashi Setsuo

Cancer research 62 ( 10 ) 2971-2975 2002.05

Research paper (scientific journal), Joint Work

Implications of peritoneal washing cytology in patients with potentially resectable pancreatic cancer

Yachida Shinchi, Fukushima Noriyoshi, Sakamoto Michiie, Matsuno Y, Kosuge T, Hirohashi Setsuo

British journal of surgery 89 ( 5 ) 573-578 2002.05

Research paper (scientific journal), Joint Work

Detection of genes expressed in primary colon cancers by in situ hybridization: overexpression of RACK 1

Saito A, Fujii G, Sato Y, Gotoh M, Sakamoto Michiie, Toda G, Hirohashi Setsuo

Molecular pathology 55 ( 1 ) 34-39 2002.02

Research paper (scientific journal), Joint Work

Overexpression of KIT in chromophobe renal cell carcinoma

Yamazaki Ken, Sakamoto Michiie, Ohta Tsutomu, Kanai Yae, Ohki Misaro, Hirohashi Setsuo

Oncogene 22 ( 6 ) 847-852 2002.02

Research paper (scientific journal), Joint Work

Overexpression of osteopontin in hepatocellular carcinoma

Gotoh, M., Sakamoto, M., Kanetaka, K., Chuuma, M. and Hirohashi, S.

Pathology International 52 ( 1 ) 19 - 24 2002.01

ISSN  1320-5463

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Human hepatocellular carcinoma (HCC) is one of the most common and aggressive malignancies. In order to identify genes involved in HCC progression, we conducted a differential display analysis and found osteopontin (OPN) to be overexpressed in HCC. OPN is known to be a secreted adhesive glycoprotein, associated with tumorigenesis and metastasis in several cancers. Quantitative polymerase chain reaction analysis of 30 HCC cases revealed the average ratio of OPN to glyceraldehyde-3-phosphate dehydrogenase in tumors to be significantly higher than that in the surrounding non-cancerous liver (4.7 +/- 1.6 vs 0.18 +/- 0.04, P = 0.0072). Immunohistochemistry confirmed the OPN protein was expressed mainly on cancer cells, and was positive in 12 of 30 HCC, most of which showed transcript overexpression. Both OPN transcript and OPN protein were significantly overexpressed in HCC with capsular infiltration, compared with HCC without capsular infiltration. Moreover, OPN-positive cancer cells were often dispersed in the periphery of cancer nodules and were adjacent to stromal cells. Although OPN overexpression was not related to vascular invasion or intrahepatic metastasis, OPN was suggested to play a role in HCC, especially in cancer-stromal interactions.

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Dysadherin, a cancer-associated cell membrane glycoprotein, down-regulates E-cadherin and promotes metastasis

Ino Yoshinori, Gotoh Masahiro, Sakamoto Michiie, Tsukagoshi Kiyomi, Hirohashi Setsuo

Proceedings of the National Academy of Sciences of the United States of America 99 ( 1 ) 365-370 2002.01

Research paper (scientific journal), Joint Work

Epidermal growth factor receptor gene amplification is correlated with laminin-5 γ2 chain expression in oral squamous cell carcinoma cell lines

Ono Yukiko, Nakanishi Yukihiro, Gotoh Masahiro, Sakamoto Michiie, Hirohashi Setsuo

Cancer letters 175 ( 2 ) 197-204 2002.01

Research paper (scientific journal), Joint Work

Overexpression of osteopontin in hepatocellular carcinoma

Gotoh Masahiro, Sakamoto Michiie, Kanetaka Kengo, Chuuma Makoto, Hirohashi Setsuko

Pathology international 52 ( 1 ) 19-24 2002.01

Research paper (scientific journal), Joint Work

Expression Profiling in Multistage Hepatocarcinogenesis:Identification of HSP70 as a Molecular Marker of Early Hepatocellular Carcinoma

Chuma Makoto, Sakamoto Michiie, Yamazaki Ken, Ohta Tsutomu, Ohki Misao, Asaka Masahiro, Hirohashi Setsuo

Hepatology 37 ( 1 ) 198-207 2002.01

Research paper (scientific journal), Joint Work

早期肝細胞癌・前癌病変の病理と自然経過，

Sakamoto Michiie

肝細胞癌治療マニュアル  2002

Research paper (scientific journal), Single Work

Ras Farnesylation Inhibitor FTI-277 Restores the E-Cadhelin/Catenin Cell Adhension System in Human Cancer Cells and Reduces Cancer Metastasis

Nam JS, Ino Yoshinori, Sakamoto Michiie, Hirohashi Setsuo

Japanese journal of cancer reseach 93   1020-1028 2002

Research paper (scientific journal), Joint Work

肝発癌の分子機構

Kanai Yae, Sakamoto Michiie

Annual Review消化器2002    113-118 2002

Research paper (scientific journal), Joint Work

肝炎と肝癌の分子病理学

Sakamoto Michiie

Molecular Medicine 1139 ( 8 )  2002

Research paper (scientific journal), Single Work

肝発癌の分子機構

Kanai Yae, Sakamoto Michiie

Annual Review消化器 2002   113-118 2002

Research paper (scientific journal), Joint Work

Overexpression of tetraspanin CO-029 in hepatocellular carcinoma

Kanetaka, K., Sakamoto, M., Yamamoto, Y., Yamasaki, S., Lanza, F., Kanematsu, T. and Hirohashi, S.

Journal of Hepatology 35 ( 5 ) 637 - 642 2001.11

ISSN  0168-8278

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Background/Aims: The molecules involved in the progression of hepatocellular carcinoma (HCC) are not fully understood. The aim of this study was to elucidate the crucial genes involved in cancer progression and metastasis.

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Involvement of c-Src in carcinoma cell motility and metastasis

Sakamoto, M., Takamura, M., Ino, Y., Miura, A., Genda, T. and Hirohashi, S.

Japanese Journal of Cancer Research 92 ( 9 ) 941 - 946 2001.09

ISSN  0910-5050

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Carcinoma cells exhibit dysfunction/dysregulation of cell adhesion systems that correlates with their abilities to migrate, invade, and metastasize. Here we show that the tyrosine kinase c-Src is required for motility and metastasis of two carcinoma cell lines. Adherent KYN-2 cells having a high level of c-Src kinase activity become scattered, extend lamellipodia, and exhibit high motility. Expression of a dominant-negative mutant form of c-Src caused formation of stress fibers and focal adhesions, and markedly reduced motility. HCT15 cells extended lamellipodia and became scattered in response to lysophosphatidic acid stimulation in parallel with transient activation of c-Src, which was inhibited by expression of a dominant-negative mutant form of c-Src or treatment with a specific Src kinase inhibitor. Furthermore, implantation of dominant-negative c-Src transfectants into the peritoneal cavity of SCID mice resulted in reduced peritoneal dissemination compared with control transfectants. These findings indicate that c-Src activation is critically involved in carcinoma cell migration and metastasis.

Inhibition of intrahepatic metastasis of human hepatocellular carcinoma by Rho-associated protein kinase inhibitor Y-27632

Takamura, M., Sakamoto, M., Genda, T., Ichida, T., Asakura, H. and Hirohashi, S.

Hepatology 33 ( 3 ) 577 - 81 2001.03

ISSN  0270-9139

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Intrahepatic metastasis is one of the most important prognostic factors for patients with hepatocellular carcinoma (HCC). Cell motility mediated by Rho- and p160 Rho-associated coiledcoil forming protein kinase (p160ROCK) signaling pathways has recently been shown to play a critical role in intrahepatic metastasis in human HCC. Furthermore, the stable introduction of dominant-negative p160ROCK into Li7 cells resulted in a reduced metastatic rate in mice with severe combined immunodeficiency (SCID). To investigate whether the specific p160ROCK inhibitor, Y-27632, could also inhibit intrahepatic metastasis, the effect of Y-27632 on the cell motility and intrahepatic metastasis of Li7 was investigated. Y-27632 markedly blocked actin reorganization and motility of Li7 cells mediated by lysophosphatidic acid (LPA). Y-27632 was administered continuously into the peritoneal cavity using a micro-osmotic pump, together with orthotopic implantation of Li7 cells into the liver of SCID mice. Phosphate-buffered saline (PBS) alone was administered as the control. The incidence of mice with metastatic nodules decreased in the Y-27632-treated group. The primary tumor volume at the site of injection was smaller in the Y-27632-treated group compared with the control group, but the difference was not statistically significant. Histologically, control tumors showed infiltrative growth into the sinusoidal area at the tumor boundary, whereas Y-27632-treated tumors showed expansive growth and low invasiveness. These findings confirm the importance of the Rho/p160ROCK signaling pathway in intrahepatic metastasis of human HCC, and indicate that Y-27632 may be useful for the prevention of intrahepatic metastasis of human HCC.

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Expression of mRNA for DNA methyltransferases and methyl-CpG-binding proteins and DNA methylation status on CpG islands and pericentromeric satellite regions during human hepatocarcinogenesis

Saito Yoshimasa, Kanai Yae, Sakamoto Michiie, Saito Hidetsugu, Ishii Hiromasa, Hirohashi Setsuo

Hepatology 33 ( 3 ) 561-568 2001.03

Research paper (scientific journal), Joint Work

Cloning and characterization of a novel gene, DRH1, down-regulated in advanced human hepatocellular carcinoma

Yamamoto, Y., Sakamoto, M., Fujii, G., Kanetaka, K., Asaka, M. and Hirohashi, S.

Clinical Cancer Research 7 ( 2 ) 297 - 303 2001.02

ISSN  1078-0432

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Few genes related to carcinogenesis and progression of hepatocellular carcinoma (HCC) have been identified to date. In the present study, we report the cloning and characterization of a novel gene, DRH1, which is frequently down-regulated in HCC. The full-length DRH1 clone contains an open reading frame of 1257 nucleotides encoding 419 amino acids. The deduced DRH1 protein shows 41% identity to VDUP1, expression of which is rapidly induced by 1,25-dihgdroxyvitamin D-3. The DRH1 gene was localized to chromosome 15, and DRH1 protein was mainly observed in the cytoplasm of transiently transfected cells. Real-time quantitative reverse transcription-PCR analysis showed that the expression level of DRH1 was reduced in 29 of 35 (83 %) HCCs compared with corresponding noncancerous liver tissue. The average (mean +/- SE) ratio of DRH1 expression level in tumor to corresponding noncancerous tissue was significantly different between well, moderately, and poorly differentiated HCCs (1.15 +/- 0.23, 0.69 +/- 0.10, and 0.19 +/- 0.04, respectively) and between HCCs without and with vascular invasion (0.93 +/- 0.16 and 0.46 +/- 0.07, respectively). These results indicate that the down-regulation of DRH1 occurs not at an early stage but rather at a late stage of HCC progression. Although the function of DRH1 protein is still unknown, our findings suggest that DRH1 is related to the progression of HCC and may provide a new prognostic factor.

特集：知っておくべき術中診断法肝癌の術中診断-病理-

Sakamoto Michiie

外科 64 ( 1 ) 32-36 2001.01

Research paper (scientific journal), Single Work

Adoptive immunotherapy to lower postsurgical recurrence rates of hepatocellular carcinoma: a randomised trial

Takayama, T., Sekine, T., Makuuchi, M., Yamasaki, S., Kosuge, T., Yamamoto, J., Shimada, K., Sakamoto, M., Hirohashi, S., Ohashi, Y. and Kakizoe, T.

Lancet 356 ( 9232 ) 802 - 807 2000.09

ISSN  0140-6736

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Background Postsurgical recurrence of hepatocellular carcinoma (HCC) is frequent and fatal. Adoptive immunotherapy is active against HCC. We assessed whether postoperative immunotherapy could lower the frequency of recurrence.
Methods Between 1992 and 1995, we did a randomised trial in which 150 patients who had undergone curative resection for HCC were assigned adoptive immunotherapy (n=76) or no adjuvant treatment (n=74). Autologous lymphocytes activated vitro with recombinant interleukin-2 and antibody to CD3 were infused five times during the first 6 months. Primary endpoints were time to first recurrence and recurrence-free survival and analyses were by intention to treat.
Findings 76 patients received 370 (97%) of 380 scheduled lymphocyte infusions (mean cell number per patient 7.1x10(10) [SD 2.1]; CD3 and HLA-DR ceils 78% [16]), and none had grade 3 or 4 adverse events. After a median follow-up of 4.4 years (range 0.2-6.7), adoptive immunotherapy decreased the frequency of recurrence by 18% compared with controls (45% [59] vs 57% [77]) and reduced the risk of recurrence by 41% (95% CI 12-60, p=0.01). Time to first recurrence in the immunotherapy group was significantly longer than that in the control group (48% [37-59] vs 33% [22-43] at 3 years, 38% [22-54] vs 22% [11-34] at 5 years; p=0.008). The immunotherapy group had significantly longer recurrence-free survival (p=0.01) and disease-specific survival (p=0.04) than the control group. Overall survival did not differ significantly between groups (p=0.09).
Interpretation Adoptive immunotherapy is a safe, feasible treatment that can lower recurrence and improve recurrence-free outcomes after surgery for HCC.

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Loss of cell-cell contact is induced by integrin-mediated cell-substratum adhesion in highly-motile and highly-metastatic hepatocellular carcinoma cells

Genda, T., Sakamoto, M., Ichida, T., Asakura, H. and Hirohashi, S.

Lab Invest 80 ( 3 ) 387 - 94 2000.03

ISSN  0023-6837

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The cadherin-mediated cell-cell adhesion system plays a critical role in normal development and morphogenesis. Inactivation of this system is thought to be responsible for cancer invasion and metastasis. A human hepatocellular carcinoma (HCC) cell line, KYN-2, was observed to have great potential for intrahepatic metastasis when orthotopically implanted into the liver of SCID mice. In vitro cultures of KYN-2 cells showed that they formed trabecular structures in suspension but lost tight cell-cell adhesion and became scattered when attached to a substratum such as collagen or fibronectin. In response to adhesion to the substratum, subcellular colocalization of E-cadherin and actin filaments were shown to be reduced, and a significant amount of alpha-catenin was dissociated from the E-cadherin-catenin complex in KYN-2 cells. These changes of cell-cell adhesion were blocked by inhibitory monoclonal antibodies against beta1 and beta5 integrins. We found that c-Src was coimmunoprecipitated with E-cadherin-catenin complex and was tyrosine-dephosphorylated and activated in the adherent cells. The tyrosine dephosphorylation of c-Src was induced by cell adhesion to the substratum and inhibited by addition of inhibitory monoclonal antibodies against beta1 and beta5 integrins. These findings indicate that integrin-mediated cell-substratum adhesion inhibits cadherin-mediated cell-cell adhesion, possibly through c-Src activation, and suggest that this cross-talk mediates transient inactivation of the cadherin system and plays an important role in intrahepatic metastasis of human HCC. Modulation of this interaction might provide a new approach to prevent metastasis and recurrence of HCC.

Cell motility mediated by rho and Rho-associated protein kinase plays a critical role in intrahepatic metastasis of human hepatocellular carcinoma

Genda, T., Sakamoto, M., Ichida, T., Asakura, H., Kojiro, M., Narumiya, S. and Hirohashi, S.

Hepatology 30 ( 4 ) 1027 - 36 1999.10

ISSN  0270-9139

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Human hepatocellular carcinoma (HCC) can invade the portal vein and metastasizes to other parts of the liver even at a relatively early stage of the disease, with less tumor spread occurring outside the liver. This intrahepatic metastasis is the main cause of liver failure and death in HCC patients. To analyze the mechanisms of intrahepatic metastasis we have constructed metastatic models using orthotopic implantation of human HCC cell lines. Five HCC cell lines formed liver tumors after injection into the livers of SCID mice, and of those 5 cell lines, Li7 and KYN-2 cells also resulted in vascular tumor thrombi and intrahepatic metastasis. These 2 cell lines had markedly higher cell motilities than the other 3 cell lines in vitro. Their motilities appeared to be Rho-mediated; serum and lysophosphatidic acid (LPA) evoked actin reorganization and motility of Li7 cells, and C3 exoenzyme exposure reduced the motility of both serum-stimulated Li7 cells and KYN-2 cells. Dominant negative and active forms of p160 Rho-associated coiled-coil forming protein kinase (p160ROCK), one of the downstream effectors of Rho, were separately and stably introduced into Li7 cells. Dominant active p160ROCK transfectants showed increased motility that was independent of serum and LPA, and dominant negative p160ROCK transfectants showed reduced motility under stimulation. Furthermore, implantation of dominant negative p160ROCK transfectants resulted in a reduced metastatic rate in vivo compared with the parent cells or a control transfectant. These findings indicate that cell motility mediated by the Rho/p160ROCK signaling pathway plays a critical role in intrahepatic metastasis of human HCC.

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Microsatellite instability associated with hepatocarcinogenesis

Kondo, Y., Kanai, Y., Sakamoto, M., Mizokami, M., Ueda, R. and Hirohashi, S.

J Hepatol 31 ( 3 ) 529 - 36 1999.09

ISSN  0168-8278

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BACKGROUND/AIMS: The biological and clinicopathological significance of microsatellite instability in hepatocellular carcinoma still remains to be determined. The aim of this study was to assess the role of microsatellite instability in hepatocarcinogenesis. METHODS: Genomic DNA extracted from 38 fresh samples of hepatocellular carcinoma was amplified by polymerase chain reaction using 29 fluorescence-labeled microsatellite markers and analyzed using a semi-automated laser scanning system. Associations between the incidence of replication error and the clinicopathological features of hepatocellular carcinoma were evaluated. Since reference DNA was extracted from corresponding fresh samples of non-cancerous liver tissue, the incidence of microsatellite instability in non-cancerous liver tissues was not assessed in this study. RESULTS: Four (11%) hepatocellular carcinomas had a replication error in one or two microsatellite markers; they were all poorly differentiated hepatocellular carcinomas. The incidence of replication error correlated significantly with the histological differentiation of the tumor (p<0.05) and with portal vein involvement (p<0.05). All four hepatocellular carcinomas with replication errors showed loss of heterozygosity in one or more of the 29 markers we examined. No replication errors were detected in six markers in the coding regions of the BAX, insulin-like growth factor II receptor, transforming growth factor-beta type II receptor, E2F-4, hMSH3 and hMSH6 genes. CONCLUSIONS: The results of this study indicate that: (1) microsatellite instability is a rare event during hepatocarcinogenesis and may be specifically associated with progression of hepatocellular carcinoma; and (2) frame-shift mutation in the above six genes is not a common mechanism involved in progression of this cancer.

Acquisition of glutamine synthetase expression in human hepatocarcinogenesis: relation to disease recurrence and possible regulation by ubiquitin-dependent proteolysis

Osada, T., Sakamoto, M., Nagawa, H., Yamamoto, J., Matsuno, Y., Iwamatsu, A., Muto, T. and Hirohashi, S.

Cancer 85 ( 4 ) 819 - 31 1999.02

ISSN  0008-543X

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BACKGROUND: The authors previously reported increased ubiquitin (Ub) immunoreactivity in hepatocellular carcinomas (HCCs) and suggested a possible correlation between changes in ubiquitinated protein levels and multistep hepatocarcinogenesis. The current study was performed to identify one of these ubiquitinated proteins (42 kDa) and to analyze the clinical significance of its accumulation. METHODS: The protein was purified using two-dimensional gel electrophoresis and identified by amino acid sequence analysis. The authors studied the expression of this protein in 101 HCCs and 23 precancerous lesions by immunohistochemical methods and in 26 HCCs by immunoblot analysis. A survival analysis was performed on patients with advanced HCC using the Kaplan-Meier method with approximate chi-square statistics for the log rank test. RESULTS: The target protein for ubiquitination was identified as glutamine synthetase (GS). Accumulation of GS was found in 19 of 49 advanced HCCs (38.8%) by immunohistochemical methods and in 9 of 16 (56.3%) by immunoblot analysis, whereas the frequency was much lower in early HCCs (12.9% and 33.3%, respectively) and precancerous lesions (4.3% by immunostaining). In the Ub immunoblot analysis of strongly GS positive specimens, an intense 42-kDa ubiquitinated band was observed. Nine of 21 (42.9%) nodule-in-nodule type HCCs showed a GS positive, high-grade component within a GS negative, low-grade component, indicating the acquisition of GS expression during progression. Among 23 patients with a single advanced HCC nodule, the relapse free survival time was significantly shorter in the GS positive group than in the GS negative group. CONCLUSIONS: The results of this study demonstrate the acquisition of GS expression during hepatocarcinogenesis and the possible regulation of GS enzyme activity by a Ub-dependent proteolytic system. Moreover, GS might play a significant role in promoting the metastatic potential of HCC.

Early hepatocellular carcinoma as an entity with a high rate of surgical cure

Takayama, T., Makuuchi, M., Hirohashi, S., Sakamoto, M., Yamamoto, J., Shimada, K., Kosuge, T., Okada, S., Takayasu, K. and Yamasaki, S.

Hepatology 28 ( 5 ) 1241 - 1246 1998.11

ISSN  0270-9139

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Early hepatocellular carcinoma (HCC) has been defined as a well-differentiated cancer containing Glisson's triad, but it remains unknown whether this lesion is curable. We prospectively studied 70 patients (enrolled from 1,172 referrals between 1982 and 1991) who had a diagnosis of a single HCC 2 cm or less in diameter (Stage T1) and who underwent curative hepatectomy and long-term follow-up (range, 0.2 to 14.3 years), Patients were eligible for surgery if they had a tumor that met the diagnostic criteria for HCC and were in Child-Pugh class A (n = 59) or B (n = 11) status. Among the 70 patients, there was 1 operative death. Based on our typing system, the tumors were assigned as early HCC (n = 15), overt HCC (n = 52), and non-HCC tumor (n = 3), The rate of microscopic regional spread was lower in early HCCs than in overt HCCs (7% vs. 42%; P =.01). The early HCC group had a longer time to recurrence than did the overt HCC group (3.9 vs. 1.7 years; P <.001) and had no local recurrence, After a median follow-up of 6.3 years, both overall survival and recurrence-free survival in the early HCC group were significantly better than those in the overt HCC group (P =.01; P =.001). In these two groups, the 5-year rates of overall survival were 93% and 54% (P =.01), and those of recurrence-free survival were 47% and 16% (P =.05), respectively; a significant survival benefit persisted over a decade (57% vs. 21%; P =.05). The early HCC group was at a lower risk of recurrence (relative risk, 0.31; 95% CI, 0.15 to 0.65; P =.002) and death (relative risk, 0.26; 95% CI, 0.09 to 0.73; P =.01) than was the overt HCC group. Early HCC is a distinct clinical entity with a high rate of surgical cure, thereby justifying its definition. It can be a lesion that corresponds to "Stage 0" cancer in other organs.

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Hyperplastic foci reflect the risk of multicentric development of human hepatocellular carcinoma

Sugitani, S., Sakamoto, M., Ichida, T., Genda, T., Asakura, H. and Hirohashi, S.

J Hepatol 28 ( 6 ) 1045 - 53 1998.06

ISSN  0168-8278

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BACKGROUND/AIMS: Identification of the risk factors of multicentric hepatocarcinogenesis is important for the clinical management of hepatocellular carcinoma. We investigated hyperplastic foci in non-cancerous liver parenchyma, and clarified their pathological features and clinical significance. METHODS: Hyperplastic foci were defined as hypercellular areas, which architecturally and cytologically resembled early hepatocellular carcinoma or adenomatous hyperplasia but did not form macroscopically detectable nodules. Surgically resected livers from 155 patients with hepatocellular carcinoma were examined histopathologically and immunohistochemically. RESULTS: Hyperplastic foci were found in 26 of 155 patients (16.8%). All the patients with hyperplastic foci had chronic liver diseases, and the incidence did not differ between those with chronic hepatitis and those with liver cirrhosis. Six of 92 (6.5%) patients with single primary hepatocellular carcinoma nodules, 8 of 42 (19.0%) with two nodules, and 12 of 21 (57.0%) with more than three nodules had hyperplastic foci. The incidence of hyperplastic foci showed a significant positive correlation with the multiplicity of hepatocellular carcinoma nodules. Immunohistochemically, hyperplastic foci were masses of proliferative hepatocytes similar to adenomatous hyperplasia and early hepatocellular carcinoma. CONCLUSIONS: Hyperplastic foci reflect the risk of multicentric hepatocarcinogenesis. Our results suggest strongly that hyperplastic foci are precursors of adenomatous hyperplasia or hepatocellular carcinoma.

Increased ubiquitin immunoreactivity in hepatocellular carcinomas and precancerous lesions of the liver

Osada, T., Sakamoto, M., Nishibori, H., Iwaya, K., Matsuno, Y., Muto, T. and Hirohashi, S.

J Hepatol 26 ( 6 ) 1266 - 73 1997.06

ISSN  0168-8278

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BACKGROUND/AIMS: Ubiquitin covalently attaches to abnormal and short-lived proteins, thus marking them for ATP-dependent proteolysis in eukaryotic cells. Increased ubiquitin immunoreactivity was recently observed immunohistochemically in human malignant tumors. To clarify the change in protein metabolism during hepatocarcinogenesis, we studied ubiquitin immunoreactivity in hepatocellular carcinomas (HCCs) and precancerous lesions using immunohistochemistry and immunoblot analysis. METHODS: A total of 72 HCCs (37 advanced, 19 early, 16 early-advanced (advanced HCC component in early HCC nodule) type HCCs) and 18 precancerous lesions (8 atypical adenomatous hyperplasias (AAHs), 10 adenomatous hyperplasias (AHs)) were studied immunohistochemically. Immunoblot analysis was also performed in advanced HCC and early HCC cases. RESULTS: Non-tumorous hepatocytes were either immunonegative or weakly stained in their nuclei. Advanced HCCs showed strong immunoreactivity in most cases, while early HCCs showed relatively weaker immunoreactivity. In 14 of 16 early-advanced type tumors, the inner portion of the nodules, which corresponds to advanced HCC, showed stronger immunoreactivity than the outer low-grade portion. In 8 of 8 AAHs and 7 of 10 AHs, positive but weak staining was found. Immunoblot analysis showed an increase in 42 kDa ubiquitinated protein(s) in 8 of 16 advanced HCC cases (50%) and in 1 of 6 early HCC cases (16.7%), as well as an increase in several other bands in tumor tissues. CONCLUSIONS: The intensity of ubiquitin staining appeared to increase in a stepwise manner from AH to advanced HCC, and the results suggest a possible correlation between changes in the ubiquitinated proteins and multistep hepatocarcinogenesis.

Reduced p21(WAF1/CIP1) expression and p53 mutation in hepatocellular carcinomas

Hui, A. M., Kanai, Y., Sakamoto, M., Tsuda, H. and Hirohashi, S.

Hepatology 25 ( 3 ) 575 - 9 1997.03

ISSN  0270-9139

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p21(WAF1/CIP1) is a universal cyclin-dependent kinase (cdk) inhibitor, the expression of which is regulated by p53-dependent and p53-independent pathways. We examined p21(WAF1/CIP1) expression in and p53 status of 21 primary hepatocellular carcinomas (HCCs) by reverse-transcriptase polymerase chain reaction (RT-PCR) and by PCR single-strand conformation polymorphism (PCR-SSCP) analysis. p21(WAF1/CIP1) messenger RNA expression was reduced markedly in 8 of 21 HCCs (38.1%) and 5 of these 8 HCCs bore p53 mutations. The relative p21(WAF1/CIP1) messenger RNA expression value of HCCs with p53 mutations (.73 +/- .13 U, n = 6) was significantly lower than that of HCCs with wild-type p53 (1.00 +/- 0.21 U, n = 14; P < .01). The p21(WAF1/CIP1) expression levels in cancerous tissues (.73 +/- .13 U) were significantly reduced in comparison with those in noncancerous tissues (.97 +/- .13 U) (P < .01) in the 6 cases with p53 mutations. These data indicate that p21(WAF1/CIP1) expression in HCCs is predominantly regulated by dependence on p53 and that reduced p21(WAF1/CIP1 expression may participate in hepatocarcinogenesis.

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E-cadherin is involved in the intrahepatic metastasis of hepatocellular carcinoma

Osada, T., Sakamoto, M., Ino, Y., Iwamatsu, A., Matsuno, Y., Muto, T. and Hirohashi, S.

Hepatology 24 ( 6 ) 1460 - 7 1996.12

ISSN  0270-9139

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In human hepatocellular carcinoma (HCC), the liver is the major target organ of metastasis, which is known as intrahepatic metastasis. To analyze the mechanism of this metastasis, we established two sublines from the human HCC cell line Li7. Subline Li7HM produced multiple liver metastasis, whereas subline Li7NM never did so after intrasplenic injection into nude mice. Two-dimensional gel electrophoresis and immunoblot analysis showed that only Li7NM expressed vimentin and lacked E-cadherin expression, indicating that this clone had undergone epithelial-mesenchymal transition. We then transfected mouse E-cadherin complementary DNA into Li7NM cells and found that the transfectant cells (EM16.21B.3) formed liver metastasis (8/16 mice) after intrasplenic injection and liver tumors (11/13 mice) after intrahepatic injection, whereas the control cell line formed no tumors. These results suggest that E-cadherin plays an important role in the process of intrahepatic metastasis of HCC.

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Numerical chromosome aberrations in hepatocellular carcinoma detected by fluorescence in situ hybridization

Ohsawa, N., Sakamoto, M., Saito, T., Kobayashi, M. and Hirohashi, S.

J Hepatol 25 ( 5 ) 655 - 62 1996.11

ISSN  0168-8278

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AIMS/METHODS: Numerical aberrations of chromosomes 7,8,9,10,12,17,18, X and Y were examined in 38 hepatocellular carcinoma specimens using centromere-specific DNA probes by the fluorescence in situ hybridization method. RESULTS: Numerical aberrations in more than one of the chromosomes examined were found in 27 of 38 (71%) specimens; 6 of 15 (40%) well-differentiated hepatocellular carcinomas; 15 of 17 (88%) moderately differentiated hepatocellular carcinomas; and all of 6 (100%) poorly differentiated hepatocellular carcinomas. Of 6 early hepatocellular carcinomas, numerical chromosome aberrations were detected in 2. The incidence of numerical chromosome aberrations was 93.8% in patients with portal vein thromboses and/or intrahepatic metastases, 52.4% without portal vein thromboses and/or intrahepatic metastases (p < 0.05), while 89.5% of patients with a tumor more than 3 cm in diameter and 50.0% with a tumor less than 3 cm in diameter had chromosome aberrations (p < 0.05). CONCLUSIONS: These results suggest that numerical chromosome aberrations start to occur in the early stage of hepatocellular carcinoma and to accumulate during tumor progression.

Inactivation of p16INK4 in hepatocellular carcinoma

Hui, A. M., Sakamoto, M., Kanai, Y., Ino, Y., Gotoh, M., Yokota, J. and Hirohashi, S.

Hepatology 24 ( 3 ) 575 - 9 1996.09

ISSN  0270-9139

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We analyzed the p16INK4 status of 6 hepatocellular carcinoma (HCC) cell lines and 32 primary HCC tumors, including 9 early-stage tumors, to determine whether p16INK4 tumor-suppressor gene inactivation participates in hepatocarcinogenesis. p16INK4 was studied at its protein level through Western blotting, at its messenger RNA (mRNA) level through reverse-transcriptase polymerase chain reaction analysis (RT-PCR) and Northern blotting, and at its genomic level through Southern blotting and PCR-single-strand conformation polymorphism analysis. The p16 protein was absent from 3 of 6 cell lines (50%) and 11 of 32 primary tumors (34%), but present in noncancerous tissues, indicating that p16INK4 is involved in hepatocarcinogenesis. Furthermore, we suggest that the p16 protein loss may contribute to the following: (1) early-stage hepatocarcinogenesis, because it was observed in 22% of early stage tumors; and (2) tumor progression, because it occurred approximately twice as often in advanced rather than in early stage tumors (40%). It was striking that neither p16INK4 homozygous deletion and mutation nor loss of p16INK4 mRNA expression were observed in HCC cell lines and primary tumors, including those specimens from which the p16 protein was absent except the Li7HM cell line, in which p16INK4 mRNA was not detected. These results suggest that p16INK4 in HCC is inactivated predominantly by posttranscriptional regulation rather than by genomic aberrations and lack of transcription.

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Early hepatocellular carcinoma: appearance at CT during arterial portography and CT arteriography with pathologic correlation

Takayasu, K., Muramatsu, Y., Furukawa, H., Wakao, F., Moriyama, N., Takayama, T., Yamasaki, S., Sakamoto, M. and Hirohashi, S.

Radiology 194 ( 1 ) 101 - 5 1995.01

ISSN  0033-8419

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PURPOSE: To use computed tomography (CT) during arterial portography (CTAP) and CT arteriography to compare the hemodynamic properties of early hepatocellular carcinoma (HCC) with those of small HCC. MATERIALS AND METHODS: Forty-four early HCCs (mean diameter, 1.5 cm) in 37 patients (26 men and 11 women aged 52-74 years; mean age, 59.2 years) were studied. CTAP was performed on 35 early HCCs, CT arteriography on 20, and both studies on 11. CTAP, CT arteriography, or both were performed on 90 small HCCs (mean diameter, 2.0 cm) in 57 patients (44 men and 13 women aged 48-71 years; mean age, 61 years). The findings for small HCC were compared with those for early HCC. RESULTS: CTAP depicted 23 early HCCs as hypoattenuating masses and 12 as isoattenuating. CT arteriography depicted 11 early HCCs as hypoattenuating masses, six as isoattenuating, and three as hyperattenuating. CTAP depicted 85 of 88 small HCCs as hypoattenuating masses and three as isoattenuating. CT arteriography depicted 13 of 14 small HCCs as hyperattenuating masses. CONCLUSION: CTAP, the standard of reference for the detection of small HCC, is not sensitive enough for the detection of early HCC.

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Radiotherapy for hepatocellular carcinoma: clinicopathological study of seven autopsy cases

Aoki, K., Okazaki, N., Okada, S., Nose, H., Yoshimori, M., Akine, Y., Egawa, S., Sakamoto, M. and Hirohashi, S.

Hepatogastroenterology 41 ( 5 ) 427 - 31 1994.10

ISSN  0172-6390

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The clinical value of radiotherapy for hepatocellular carcinoma (total tumor dose: 50-70 Gy) was evaluated in seven autopsied patients with nine tumors. A partial response as defined by the WHO criteria for chemotherapy was observed in three tumors (33%). Progressive and marked shrinkage of the noncancerous liver tissue within the irradiated field was also observed in all cases. Histological examination revealed viable cancer cells in all cases. These results indicate that local radiotherapy applying 50-70 Gy achieved some clinical benefit, but was not capable of curing hepatocellular carcinoma.

Predictive factors for postoperative recurrence of hepatocellular carcinoma

Okada, S., Shimada, K., Yamamoto, J., Takayama, T., Kosuge, T., Yamasaki, S., Sakamoto, M. and Hirohashi, S.

Gastroenterology 106 ( 6 ) 1618 - 24 1994.06

ISSN  0016-5085

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BACKGROUND/AIMS: The survival rate for hepatocellular carcinoma after hepatectomy remains unsatisfactorily low because of the high recurrence rate. The purpose of this study was to elucidate predictive factors for postoperative recurrence of hepatocellular carcinoma. METHODS: Univariate analysis and a multiple regression model were used to retrospectively determine the factors potentially related to recurrence in 98 patients with hepatocellular carcinoma who had undergone curative hepatic resection. RESULTS: DNA diploidy, absence of alcohol abuse, and absence of vascular invasion were determined to be independent favorable predictive factors using a multivariate analysis with Cox's proportional hazards model. A predictive index was developed using these three factors according to the following equation: 1.486 x (O = DNA diploidy or 1 = DNA aneuploidy) + 1.138 x (O = absence or 1 = presence of alcohol abuse) + 0.946 x (O = absence or 1 = presence of vascular invasion). This index was used to classify the patients into two groups with good or poor prognosis. The median time for disease recurrence for the two groups was 24.1 and 9.2 months, respectively (P < 0.001). CONCLUSIONS: The results may be useful in the determination of treatment strategies and postoperative follow-up schedules and in the design and analysis of future clinical trials of therapy for hepatocellular carcinoma.

Phenotype Changes in Tumor Vessels Associated with the Progression of Hepatocellular-Carcinoma

Sakamoto, M., Ino, Y., Fujii, T. and Hirohashi, S.

Japanese Journal of Clinical Oncology 23 ( 2 ) 98 - 104 1993.04

ISSN  0368-2811

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Human hepatocellular carcinoma (HCC) is characterized by hypervascularity and tumor staining in angiograms, and tumor angiogenesis is considered indispensable for tumor growth. HCC is also characterized by an obvious multistage process of tumor progression. To find out in which stage of human hepatocarcinogenesis angiogenesis occurs, we have carried out a pathological study, of the phenotypic changes in tumor vessels taken from surgically resected liver tumors showing each step of the progression. Eleven early advanced HCCs (advanced HCC component in early HCC nodule, eAd HCC), seven early HCCs (eHCC) and six adenomatous hyperplasias (AH), the non-tumorous liver surrounding each and five normal livers were studied by lectin histochemistry and immunohistochemistry. The sinusoidal endothelial cells from the non-tumorous liver were shown to be negative for UEA-I (Ulex europaeus I), but the endothelial cells from the sinusoidal tumor vessels in advanced HCC components were strongly positive. In AH and eHCC, half the tumors were negative and the other half focally positive. In the early HCC lesion of eAdHCC, the rate of positivity for UEA-I was a little higher than in AH and eHCC but lower than in advanced HCC lesions in eAdHCC. Immunohistochemically, laminin was not detected in the sinusoids in the non-tumorous liver or in the sinusoidal tumor vessels in AH and eHCC. In eAdHCC, however, two early HCC lesions and four advanced HCC lesions were positive. An immunohistochemical examination for muscle actin revealed an increase in arterial tumor vessels in six advanced HCC lesions and one early HCC lesion of eAdHCC. The results indicate the emergence of UEA-I-positive sinusoidal tumor vessels to be most pronounced during the progression from eHCC to advanced HCC, and laminin-positive sinusoidal tumor vessels or actin-positive arterial tumor vessels to emerge mainly in advanced HCC.

Pathology and DNA cytophotometry of small hepatocellular carcinoma with a nodule-in-nodule appearance--evidence for stepwise progression of hepatocellular carcinoma

Rim, K. S., Sakamoto, M., Watanabe, H., Matsuno, Y., Nakanishi, Y., Mukai, K. and Hirohashi, S.

Jpn J Clin Oncol 23 ( 1 ) 26 - 33 1993.02

ISSN  0368-2811

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Among 92 surgically resected human hepatocellular carcinomas (HCCs) < or = 3 cm in diameter, 23 tumors (25%) grossly showed a nodule-in-nodule appearance indicating stepwise progression of HCC. The central nodules showed destructive growth of HCC, which was less differentiated than the surrounding area histologically, while the surrounding area showed growth of very-well-differentiated HCC with no, or only minimal, destruction of the underlying liver structures. Previously, we proposed the term "early HCC" for HCCs without definite destructive growth, and the nodule-in-nodule lesions described here are considered to be in the transitional stage from early HCC to advanced HCC, and are therefore named early advanced HCC (eAd HCC). DNA cytophotometry was performed in 12 cases of eAd HCC, in which the early HCC component, advanced HCC component and non-tumorous liver tissue showing chronic hepatitis or cirrhosis were analyzed separately. The early HCC component showed a diploid pattern in eight of the 12 cases. The advanced HCC component showed an aneuploid peak in seven cases, and in three of these the peak was detected only in the advanced HCC component and not in the early HCC component. The mean nuclear DNA content was significantly increased from non-tumorous liver to the early HCC component and from the early HCC component to the advanced HCC component. Polyploid cells containing more than 4.8C DNA were exceptional in non-tumorous liver, but were detectable in the early HCC component and increased in number in the advanced HCC component. These findings suggest that DNA instability may have an important role to play in the subclonal progression of human HCC.

Early Stages of Multistep Hepatocarcinogenesis - Adenomatous Hyperplasia and Early Hepatocellular-Carcinoma

Sakamoto, M., Hirohashi, S. and Shimosato, Y.

Human Pathology 22 ( 2 ) 172 - 178 1991.02

ISSN  0046-8177

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Growth and spread of hepatocellular carcinoma. A review of 240 consecutive autopsy cases

Yuki, K., Hirohashi, S., Sakamoto, M., Kanai, T. and Shimosato, Y.

Cancer 66 ( 10 ) 2174 - 9 1990.11

ISSN  0008-543X

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All 240 consecutive cases of hepatocellular carcinoma (HCC) that underwent autopsy at the National Cancer Center Hospital (Tokyo, Japan) between September 1962 and August 1986 were reviewed. Among these cases, 162, for which photographs of cut surfaces of the primary tumors were available, were grossly classified using a combination of both Eggel's classification and our own into three major types, i.e., nodular, massive, and diffuse as described by Eggel (Eggel H, Beitr Pathol Anat 1901; 30:506-604), and three subgroups of nodular type, i.e., single nodular type (type 1), single nodular type with extranodular growth (type 2), and contiguous multinodular type (type 3) by our classification (Kanai T et al., Cancer 1987; 60:810-819). Seventy-eight cases were classified as nodular type, comprising seven cases of type 1, 61 cases of type 2, and ten cases of type 3. Sixty-seven and 17 cases were classified as massive and diffuse type, respectively. Of the 78 nodular-type tumors, 59 measured less than 10 cm, whereas 64 of 67 massive-type tumors were 10 cm or more in size. The incidence of intrahepatic and extrahepatic tumor spread of HCC was significantly higher for tumors measuring more than 5 cm. As to the relationship between macroscopic type and tumor spread, the frequency of spread was lowest for type 1 tumors, and high for the other types. Intrahepatic metastasis was detected in 28.6% of type 1, 93.4% of type 2, 100% of type 3, and 98.5% of massive-type tumors. Lymph node metastasis was detected in 14.3% of type 1, 24.6% of type 2, 70% of type 3, 38.8% of massive-type and 52.9% of diffuse-type tumors. Hematogenous extrahepatic metastasis was detected in 14.3% of type 1, 47.5% of type 2, 70% of type 3, 74.6% of massive-type and 82.4% of diffuse-type tumors. It appears that not only primary tumor size but also its macroscopic type has an important influence on the growth and spread of HCC.

Malignant transformation of adenomatous hyperplasia to hepatocellular carcinoma

Takayama, T., Makuuchi, M., Hirohashi, S., Sakamoto, M., Okazaki, N., Takayasu, K., Kosuge, T., Motoo, Y., Yamazaki, S. and Hasegawa, H.

Lancet 336 ( 8724 ) 1150 - 3 1990.11

ISSN  0140-6736

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To clarify the course of adenomatous hyperplasia (AH) of the liver, 17 patients with 20 biopsy-proven AH nodules were followed clinically for 1-5 years. At the initial biopsy the mean nodular diameter was 10 (SD 4) mm and the relative cellularity [( mean cellularity of AH divided by mean parenchymal cellularity] x 100) 141 (27). The criteria for diagnosis of malignant transformation of AH were both a doubling of nodular volume and changes on imaging. Between 6 and 50 months after biopsy, 9 of the 18 nodules which could still be accurately identified met the criteria for transformation; histological proof of hepatocellular carcinoma (HCC) was obtained later for 7 of these 9 nodules. The product of diameter and cellularity (transformation index) was the strongest predictor of the time to transformation. 9 AH nodules did not undergo transformation--7 did not meet one or both criteria and 2 became undetectable by imaging. Because of the high risk of malignant transformation, it can be concluded that AH is an absolute precursor of HCC. It should therefore be treated as a potential malignant disorder.

Multicentric independent development of hepatocellular carcinoma revealed by analysis of hepatitis B virus integration pattern

Sakamoto, M., Hirohashi, S., Tsuda, H., Shimosato, Y., Makuuchi, M. and Hosoda, Y.

Am J Surg Pathol 13 ( 12 ) 1064 - 7 1989.12

ISSN  0147-5185

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Two separate nodular lesions of hepatocellular carcinoma (HCC) were surgically removed from a patient with positive serum hepatitis B surface antigen. Southern blot analysis of DNA from the two separate HCC lesions showed different restriction patterns of integrated hepatitis B virus DNA, indicating their independent origins. The two lesions were composed mainly of poorly differentiated HCC; however, well-differentiated HCC was also observed in the periphery of the lesions. This histological finding is consistent with the multistep and independent development of HCC in each site of the liver.

Increasing incidence of hepatocellular carcinoma possibly associated with non-A, non-B hepatitis in Japan, disclosed by hepatitis B virus DNA analysis of surgically resected cases

Sakamoto, M., Hirohashi, S., Tsuda, H., Ino, Y., Shimosato, Y., Yamasaki, S., Makuuchi, M., Hasegawa, H., Terada, M. and Hosoda, Y.

Cancer Res 48 ( 24 Pt 1 ) 7294 - 7 1988.12

ISSN  0008-5472

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At the National Cancer Center Hospital in Japan, the total number of surgically treated hepatocellular carcinomas (HCCs) has been increasing steadily and rapidly over the last 10 years, whereas the number of cases positive for hepatitis B surface antigen in sera (HBsAg) has remained almost stable. Thus, the relative percentage of HCC cases with serum HBsAg has shown a marked decrease. In order to examine whether this increased proportion of HBsAg-seronegative patients carries hepatitis B virus (HBV) DNA in the liver, we extracted DNA from the formalin-fixed and paraffin-embedded cancerous and noncancerous liver tissues of 79 patients with HCC. The HCCs examined included 49 specimens resected during a period from 1970 to 1980 and 30 resected in 1986 and 1987. We were able to detect reliably the presence of HBV DNA by dot-blot hybridization. The presence of HBV DNA in liver tissues showed a good correlation with positivity for serum HBsAg in both examined groups. In total, HBV DNA was detected in 81% (21 of 26) of HBsAg-seropositive cases and in only 8% (4 of 53) of HBsAg-seronegative cases, indicating that the increased number of HBsAg-seronegative cases had no HBV involvement. Among these HBsAg-seronegative HCC patients, 89.7% showed a histology of cirrhosis or chronic active hepatitis in the noncancerous liver and 29.1% had a history of blood transfusion. These results suggest an increasing incidence of non-A, non-B hepatitis-associated HCCs in Japan and the possible transmission of factors by means other than blood transfusion.