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Papers - KANAI Yae

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Molecular subtypes of lung adenocarcinoma present distinct immune tumor microenvironments

Fukuda H., Arai K., Mizuno H., Nishito Y., Motoi N., Arai Y., Hiraoka N., Shibata T., Sonobe Y., Kayukawa Y., Hashimoto E., Takahashi M., Fujii E., Maruyama T., Kuwabara K., Nishizawa T., Mizoguchi Y., Yoshida Y., Watanabe S.i., Yamashita M., Kitano S., Sakamoto H., Nagata Y., Mitsumori R., Ozaki K., Niida S., Kanai Y., Hirayama A., Soga T., Tsukada K., Yabuki N., Shimada M., Kitazawa T., Natori O., Sawada N., Kato A., Yoshida T., Yasuda K., Ochiai A., Tsunoda H., Aoki K.

Cancer Science (Cancer Science) 115 ( 6 ) 1763 - 1777 2024.06

ISSN 13479032

Overcoming resistance to immune checkpoint inhibitors is an important issue in patients with non-small-cell lung cancer (NSCLC). Transcriptome analysis shows that adenocarcinoma can be divided into three molecular subtypes: terminal respiratory unit (TRU), proximal proliferative (PP), and proximal inflammatory (PI), and squamous cell carcinoma (LUSQ) into four. However, the immunological characteristics of these subtypes are not fully understood. In this study, we investigated the immune landscape of NSCLC tissues in molecular subtypes using a multi-omics dataset, including tumor-infiltrating leukocytes (TILs) analyzed using flow cytometry, RNA sequences, whole exome sequences, metabolomic analysis, and clinicopathologic findings. In the PI subtype, the number of TILs increased and the immune response in the tumor microenvironment (TME) was activated, as indicated by high levels of tertiary lymphoid structures, and high cytotoxic marker levels. Patient prognosis was worse in the PP subtype than in other adenocarcinoma subtypes. Glucose transporter 1 (GLUT1) expression levels were upregulated and lactate accumulated in the TME of the PP subtype. This could lead to the formation of an immunosuppressive TME, including the inactivation of antigen-presenting cells. The TRU subtype had low biological malignancy and “cold” tumor-immune phenotypes. Squamous cell carcinoma (LUSQ) did not show distinct immunological characteristics in its respective subtypes. Elucidation of the immune characteristics of molecular subtypes could lead to the development of personalized immune therapy for lung cancer. Immune checkpoint inhibitors could be an effective treatment for the PI subtype. Glycolysis is a potential target for converting an immunosuppressive TME into an antitumorigenic TME in the PP subtype.

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Aberrant cell adhesiveness due to DNA hypermethylation of KLF11 in papillary urothelial carcinomas

Tsumura K., Fujimoto M., Tian Y., Kawahara T., Fujimoto H., Maeshima A.M., Nakagawa T., Kume H., Yoshida T., Kanai Y., Arai E.

Experimental and Molecular Pathology (Experimental and Molecular Pathology) 137 104908 2024.06

ISSN 00144800

Purpose: The aim of this study was to clarify DNA methylation profiles determining the clinicopathological diversity of urothelial carcinomas. Methods: Genome-wide DNA methylation analysis was performed using the Infinium HumanMethylation450 BeadChip in 46 paired samples of non-cancerous urothelium (N) and corresponding cancerous tissue (T), and 26 samples of normal control urothelium obtained from patients without urothelial carcinomas (C). For genes of interest, correlation between DNA methylation and mRNA expression was examined using the Cancer Genome Atlas database. In addition, the role of a selected target for cancer-relevant endpoints was further examined in urothelial carcinoma cell lines. Results: The genes showing significant differences in DNA methylation levels between papillary carcinomas and more aggressive non-papillary (nodular) carcinomas were accumulated in signaling pathways participating in cell adhesion and cytoskeletal remodeling. Five hundred ninety-six methylation sites showed differences in DNA methylation levels between papillary and nodular carcinomas. Of those sites, that were located in CpG-islands around transcription start site, 5′-untranslated region or 1st exon, 16 genes exhibited inverse correlations between DNA methylation and mRNA expression levels. Among the latter, only the KLF11 gene showed papillary T sample-specific DNA hypermethylation in comparison to C and N samples. The DNA methylation levels of KLF11 were not significantly different between T samples and N samples or T samples and C samples for patients with papillo-nodular or nodular carcinomas. Knockdown experiments using the urothelial carcinoma cell lines HT1376 and 5637, which are considered models for papillary carcinoma, revealed that KLF11 participates in altering the adhesiveness of cells to laminin-coated dishes, although cell growth was not affected. Conclusion: These data indicate that DNA hypermethylation of KLF11 may participate in the generation of papillary urothelial carcinomas through induction of aberrant cancer cell adhesion to the basement membrane.

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Molecular pathological approach to cancer epigenomics and its clinical application

Kanai Y.

Pathology International (Pathology International) 74 ( 4 ) 167 - 186 2024.04

ISSN 13205463

Careful microscopic observation of histopathological specimens, accumulation of large numbers of high-quality tissue specimens, and analysis of molecular pathology in relation to morphological features are considered to yield realistic data on the nature of multistage carcinogenesis. Since the morphological hallmark of cancer is disruption of the normal histological structure maintained through cell−cell adhesiveness and cellular polarity, attempts have been made to investigate abnormalities of the cadherin-catenin cell adhesion system in human cancer cells. It has been shown that the CDH1 tumor suppressor gene encoding E-cadherin is silenced by DNA methylation, suggesting that a “double hit” involving DNA methylation and loss of heterozygosity leads to carcinogenesis. Therefore, in the 1990s, we focused on epigenomic mechanisms, which until then had not received much attention. In chronic hepatitis and liver cirrhosis associated with hepatitis virus infection, DNA methylation abnormalities were found to occur frequently, being one of the earliest indications that such abnormalities are present even in precancerous tissue. Aberrant expression and splicing of DNA methyltransferases, such as DNMT1 and DNMT3B, was found to underlie the mechanism of DNA methylation alterations in various organs. The CpG island methylator phenotype in renal cell carcinoma was identified for the first time, and its therapeutic targets were identified by multilayer omics analysis. Furthermore, the DNA methylation profile of nonalcoholic steatohepatitis (NASH)-related hepatocellular carcinoma was clarified in groundbreaking studies. Since then, we have developed diagnostic markers for carcinogenesis risk in NASH patients and noninvasive diagnostic markers for upper urinary tract cancer, as well as developing a new high-performance liquid chromatography-based diagnostic system for DNA methylation diagnosis. Research on the cancer epigenome has revealed that DNA methylation alterations occur from the precancerous stage as a result of exposure to carcinogenic factors such as inflammation, smoking, and viral infections, and continuously contribute to multistage carcinogenesis through aberrant expression of cancer-related genes and genomic instability. DNA methylation alterations at the precancerous stages are inherited by or strengthened in cancers themselves and determine the clinicopathological aggressiveness of cancers as well as patient outcome. DNA methylation alterations have applications as biomarkers, and are expected to contribute to diagnosis, as well as preventive and preemptive medicine.

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特集ゲノム医学を外科診療に活かす! ゲノム医療の最新動向ゲノム解析に適した手術検体の取り扱い

金井弥栄

臨床外科 (株式会社医学書院) 79 ( 2 ) 138 - 143 2024.02

ISSN 03869857

Clinicopathological significance of androgen receptor expression in extramammary Paget disease: An analysis of 92 patients

Kuramoto J, Kobayashi K, Hirai I, Nakamura Y, Funakoshi T, Kanai Y

Pathology Research and Practice (Pathology Research and Practice) 249 154775 2023.09

ISSN 03440338

Extramammary Paget disease (EMPD) is a rare cutaneous malignant neoplasm arising in apocrine gland-rich areas. Although – like normal apocrine glands – EMPD frequently expresses androgen receptor (AR), the clinical significance of AR expression remains unclear. The present study investigated the clinicopathological impact of AR expression in EMPD. Immunohistochemistry for AR was performed in a retrospective cohort of 92 EMPD patients with 108 EMPD lesions, including 102 primary lesions, five lymph node [LN] metastases and one local recurrence. The total AR staining score was calculated as staining intensity score (IS 0–3) × positive-cell percentage score (PS 1–4). Expression levels were graded as Grade 1 (scores 0 and 1), Grade 2 (scores 2–4), and Grade 3 (scores 6–12). Higher expression grade was correlated with tumor thickness (P = 0.011), LN metastasis (P = 0.008), and higher EMPD stage (P = 0.023). Grade 1 EMPDs did not invade into the dermis and did not generate metastatic and/or recurrent lesions, whereas only Grade 2 or 3 EMPDs did so. AR expression in invasive components was significantly higher (P = 0.023) than in non-invasive components remaining within the epidermis. AR expression was further elevated in metastatic and/or recurrent lesions relative to locally invasive lesions (P = 0.014). These results clearly indicate that increased AR expression is associated with malignant progression of EMPD and that androgen blockade might be an effective therapy. Furthermore, AR expression assessed by immunohistochemistry may have potential for prediction of LN metastasis and local recurrence in EMPD.

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Tumor-infiltrating Leukocyte Profiling Defines Three Immune Subtypes of NSCLC with Distinct Signaling Pathways and Genetic Alterations.

Aoki K, Nishito Y, Motoi N, Arai Y, Hiraoka N, Shibata T, Sonobe Y, Kayukawa Y, Hashimoto E, Takahashi M, Fujii E, Nishizawa T, Fukuda H, Ohashi K, Arai K, Mizoguchi Y, Yoshida Y, Watanabe SI, Yamashita M, Kitano S, Sakamoto H, Nagata Y, Mitsumori R, Ozaki K, Niida S, Kanai Y, Hirayama A, Soga T, Maruyama T, Tsukada K, Yabuki N, Shimada M, Kitazawa T, Natori O, Sawada N, Kato A, Yoshida T, Yasuda K, Mizuno H, Tsunoda H, Ochiai A

Cancer research communications (Cancer Research Communications) 3 ( 6 ) 1026 - 1040 2023.06

Resistance to immune checkpoint blockade remains challenging in patients with non–small cell lung cancer (NSCLC). Tumor-infiltrating leukocyte (TIL) quantity, composition, and activation status profoundly influence responsiveness to cancer immunotherapy. This study examined the immune landscape in the NSCLC tumor microenvironment by analyzing TIL profiles of 281 fresh resected NSCLC tissues. Unsupervised clustering based on numbers and percentages of 30 TIL types classified adenocarcinoma (LUAD) and squamous cell carcinoma (LUSQ) into the cold, myeloid cell–dominant, and CD8+ T cell–dominant subtypes. These were significantly correlated with patient prognosis; the myeloid cell subtype had worse outcomes than the others. Integrated genomic and transcriptomic analyses, including RNA sequencing, whole-exome sequencing, T-cell receptor repertoire, and metabolomics of tumor tissue, revealed that immune reaction–related signaling pathways were inactivated, while the glycolysis and K-ras signaling pathways activated in LUAD and LUSQ myeloid cell subtypes. Cases with ALK and ROS1 fusion genes were enriched in the LUAD myeloid subtype, and the frequency of TERT copy-number variations was higher in LUSQ myeloid subtype than in the others. These classifications of NSCLC based on TIL status may be useful for developing personalized immune therapies for NSCLC.

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肺腺がんのDNAメチル化プロファイルに対する免疫微小環境の影響

河原徹, 藤本真央, 下田将之, 淺村尚生, 金井弥栄, 新井恵吏

日本病理学会会誌 ((一社)日本病理学会) 112 ( 1 ) 329 - 329 2023.03

ISSN 0300-9181

New horizons for cancer research driven by histopathological approaches

Enomoto, A, Kanai, Y

CANCER SCIENCE 114 919 - 919 2023.02

ISSN 1347-9032

Epigenetic profile of Japanese supercentenarians: a cross-sectional study.

Komaki S, Nagata M, Arai E, Otomo R, Ono K, Abe Y, Ohmomo H, Umekage S, Shinozaki NO, Hachiya T, Sutoh Y, Otsuka-Yamasaki Y, Arai Y, Hirose N, Yoneyama A, Okano H, Sasaki M, Kanai Y, Shimizu A

The lancet. Healthy longevity (The Lancet Healthy Longevity) 4 ( 2 ) e83 - e90 2023.02

ISSN 2666-7568

BACKGROUND: Centenarians and supercentenarians with exceptional longevity are excellent models for research towards improvements of healthy life expectancy. Extensive research regarding the maintenance and reduction of epigenetic age has provided insights into increasing healthy longevity. To this end, we explored the epigenetic signatures reflecting hallmarks of exceptional healthy longevity, including avoidance of age-related diseases and cognitive functional decline. METHODS: In this cross-sectional study, we enrolled Japanese non-centenarians (eligible participants aged 20-80 years) from the Tohoku Medical Megabank Community-Based Cohort Study and centenarians and supercentenarians (aged 101-115 years) from the Tokyo Centenarian Study and the Japanese Semi-supercentenarian Study. We assessed participants' whole-blood DNA methylation profiles and then developed sex-specific and non-specific first-generation epigenetic clocks by elastic net regression, calculated individuals' epigenetic ages, and assessed their age acceleration. We also screened for age-related CpG sites in non-centenarians by epigenome-wide linear regression analyses and ANOVA. We subsequently investigated which CpG sites in centenarians and supercentenarians had DNA methylation patterns following the age-related findings obtained from non-centenarians and which did not. We further characterised CpG sites with hypermethylation or hypomethylation in the centenarians and supercentenarians using enrichment and protein-protein interaction network analyses. FINDINGS: We enrolled 421 non-centenarians (231 [55%] women and 190 [45%] men; age range 20-78 years), recruited between May 20, 2013, and March 31, 2016, and 94 centenarians and supercentenarians (66 women [70%] and 28 [30%] men; age range 101-115 years), recruited between Jan 20, 2001, and April 17, 2018. Non-sex-specific epigenetic clock showed the highest accuracy (r=0·96) based on which centenarians and supercentenarians had negative epigenetic age acceleration. Epigenome-wide association analyses further showed that centenarians and supercentenarians had younger-than-expected epigenetic states (DNA methylation profiles similar to those of non-centenarians) for 557 CpG sites enriched in cancer-related and neuropsychiatric-related genes, whereas these individuals had advanced (or older) epigenetic states for 163 CpG sites represented by genes related to TGF-β signalling, which is involved in anti-inflammatory responses and known to contribute to healthy ageing. INTERPRETATION: These results indicate that exceptionally healthy longevity depends not only on maintaining young epigenetic states but also on advanced states of specific epigenetic regions. FUNDING: The Japan Agency for Medical Research and Development, KDDI Research, and Keio University. TRANSLATION: For the Japanese translation of the abstract see Supplementary Materials section.

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Prospective exosome-focused translational research for afatinib (EXTRA) study of patients with nonsmall cell lung cancer harboring EGFR mutation: an observational clinical study

Takata S, Morikawa K, Tanaka H, Itani H, Ishihara M, Horiuchi K, Kato Y, Ikemura S, Nakagawa H, Nakahara Y, Seki Y, Bessho A, Takahashi N, Hayashi K, Endo T, Takeyama K, Maekura T, Takigawa N, Kawase A, Endoh M, Nemoto K, Kishi K, Soejima K, Okuma Y, Yoshimura K, Saigusa D, Kanai Y, Ueda K, Togashi A, Matsutani N, Seki N

Therapeutic Advances in Medical Oncology (Therapeutic Advances in Medical Oncology) 15 17588359231177021 2023.01

ISSN 17588340

Background: The exosome-focused translational research for afatinib (EXTRA) study is the first trial to identify novel predictive biomarkers for longer treatment efficacy of afatinib in patients with epidermal growth factor receptor (EGFR) mutation-positive nonsmall cell lung cancer (NSCLC) via a comprehensive association study using genomic, proteomic, epigenomic, and metabolomic analyses. Objectives: We report details of the clinical portion prior to omics analyses. Design: A prospective, single-arm, observational study was conducted using afatinib 40 mg/day as an initial dose in untreated patients with EGFR mutation-positive NSCLC. Dose reduction to 20 mg every other day was allowed. Methods: Progression-free survival (PFS), overall survival (OS), and adverse events (AEs) were evaluated. Results: A total of 103 patients (median age 70 years, range 42–88 years) were enrolled from 21 institutions in Japan between February 2017 and March 2018. After a median follow-up of 35.0 months, 21% remained on afatinib treatment, whereas 9% had discontinued treatment because of AEs. The median PFS was 18.4 months, with a 3-year PFS rate of 23.3%. The median afatinib treatment duration in patients with final doses of 40 (n = 27), 30 (n = 23), and 20 mg/day (n = 35), and 20 mg every other day (n = 18) were 13.4, 15.4, 18.8, and 18.3 months, respectively. The median OS was not reached, with a 3-year OS rate of 58.5%. The median OS in patients who did (n = 25) and did not (n = 78) receive osimertinib during the entire course of treatment were 42.4 months and not reached, respectively (p = 0.654). Conclusions: As the largest prospective study in Japan, this study confirmed favorable OS following first-line afatinib in patients with EGFR mutation-positive NSCLC in a real-world setting. Further analysis of the EXTRA study is expected to identify novel predictive biomarkers for afatinib. Trial registration: UMIN-CTR identifier (UMIN000024935, https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr_his_list.cgi?recptno=R000028688

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DNA methylation alterations of ADCY5, MICAL2, and PLEKHG2 during the developmental stage of cryptogenic hepatocellular carcinoma

Makiuchi S, Tian Y, Fujimoto M, Kuramoto J, Tsuda N, Ojima H, Gotoh M, Hiraoka N, Yoshida T, Kanai Y, Arai E

Hepatology Research (Hepatology Research) 54 ( 3 ) 284 - 299 2023

ISSN 13866346

Aim: The aim of this study was to clarify the significance of DNA methylation alterations of cryptogenic hepatocellular carcinomas (HCCs). Methods: Using the Infinium assay, we performed genome-wide DNA methylation analysis of 250 liver tissue samples, including noncancerous liver tissue (U-N) and corresponding cancerous tissue (U-T) from patients with cryptogenic HCC without a history of excessive alcohol use and hepatitis virus infection, and whose U-N samples showed no remarkable histological features (no microscopic evidence of simple steatosis, any form of hepatitis including nonalcoholic steatohepatitis, or liver cirrhosis). Results: We identified 3272 probes that showed significant differences of DNA methylation levels between U-N and normal liver tissue samples from patients without HCC, indicating that a distinct DNA methylation profile had already been established at the precancerous U-N stage. U-Ns have a DNA methylation profile differing from that of noncancerous liver tissue of patients with nonalcoholic steatohepatitis-related, viral hepatitis-related, and alcoholic liver disease-related HCCs. Such DNA methylation alterations in U-Ns were inherited by U-Ts. The U-Ns showed DNA methylation alteration of ADCY5, resulting in alteration of its mRNA expression, whereas noncancerous liver tissue of patients with nonalcoholic steatohepatitis-, viral hepatitis-, or alcoholic liver disease-related HCCs did not. DNA methylation levels of MICAL2 and PLEKHG2 in U-Ts were correlated with larger tumor diameter and portal vein involvement, respectively. Conclusions: U-N-specific DNA hypermethylation of ADCY5 may have significance, even from the precancerous stage in liver showing no remarkable histological features. DNA hypomethylation of MICAL2 and PLEKHG2 may determine the clinicopathological features of cryptogenic HCC.

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Single-cell analysis reveals a CD4+ T cell cluster that correlates with PD-1 blockade efficacy.

H Kagamu, S Yamasaki, S Kitano, O Yamaguchi, A Mouri, A Shiono, ...

Cancer Research, CAN-22 (Cancer research) 82 ( 24 ) 4641 - 4653 2022.10

ISSN 0008-5472

CD4+ T-cell immunity helps clonal proliferation, migration, and cancer cell killing activity of CD8+ T cells and is essential in antitumor immune responses. To identify CD4+ T-cell clusters responsible for antitumor immunity, we simultaneously analyzed the naïve-effector state, Th polarization, and T-cell receptor clonotype based on single-cell RNA-sequencing data. Unsupervised clustering analysis uncovered the presence of a new CD4+ T-cell metacluster in the CD62Llow CD4+ T-cell subpopulation, which contained multicellular clonotypes associated with efficacy of programmed death-ligand 1 (PD-1) blockade therapy. The CD4+ T-cell metacluster consisted of CXCR3+CCR4-CCR6+ and CXCR3-CCR4-CCR6+ cells and was characterized by high expression of IL7 receptor and TCF7. The frequency of these cells in the peripheral blood significantly correlated with progression-free survival and overall survival of patients with lung cancer after PD-1 blockade therapy. In addition, the CD4+ metacluster in the peripheral blood correlated with CD4+ T-cell infiltration in the tumor microenvironment, whereas peripheral Th1 correlated with local CD8+ T-cell infiltration. Together, these findings suggest that CD62Llow CCR4-CCR6+ CD4+ T cells form a novel metacluster with predictive potential of the immune status and sensitivity to PD-1 blockade, which may pave the way for personalized antitumor immunotherapy strategies for patients. SIGNIFICANCE: The identification of a new CD4+ T-cell metacluster that corresponds with immune status could guide effective tumor treatment by predicting response to immunotherapy using peripheral blood samples from patients.

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The Anterior Eye Chamber as a Visible Medium for In Vivo Tumorigenicity Tests

E Inagaki, E Arai, S Hatou, T Sayano, H Taniguchi, K Negishi, Y Kanai, ...

Stem Cells Translational Medicine (Stem cells translational medicine) 11 ( 8 ) 841 - 849 2022.06

Accepted, ISSN 2157-6564

Pluripotent stem cell (PSC)-based cell therapies have increased steadily over the past few years, and assessing the risk of tumor formation is a high priority for clinical studies. Current in vivo tumorigenesis studies require several months and depend strongly on the site of grafting. In this study, we report that the anterior eye chamber is preferable to the subcutaneous space for in vivo tumorigenesis studies for several reasons. First, cells can easily be transplanted into the anterior chamber and monitored in real-time without sacrificing the animals due to the transparency of the cornea. Second, tumor formation is faster than with the conventional subcutaneous method. The median tumor formation time in the subcutaneous area was 18.50 weeks (95% CI 10.20-26.29), vs. 4.0 weeks (95% CI 3.34-.67) in the anterior chamber (P = .0089). When hiPSCs were spiked with fibroblasts, the log10TPD50 was 3.26, compared with 4.99 when hiPSCs were transplanted without fibroblasts. There was more than a 40-fold difference in the log10TPD50 values with fibroblasts. Furthermore, the log10TPD50 for HeLa cells was 1.45 and 100% of animals formed tumors at a concentration greater than 0.1%, indicating that the anterior chamber tumorigenesis assays can be applied for cancer cell lines as well. Thus, our method has the potential to become a powerful tool in all areas of tumorigenesis studies and cancer research.

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Lymphomatoid gastropathy/NK-cell enteropathy involving the stomach and intestine

M Nakajima, M Shimoda, K Takeuchi, A Dobashi, T Kanai, Y Kanai, ...

Journal of Clinical and Experimental Hematopathology, 21032 (The Japanese Society for Lymphoreticular Tissue Research) advpub ( 0 ) 114 - 118 2022.04

Accepted, ISSN 1346-4280

<p>Lymphomatoid gastropathy (LyGa)/natural killer (NK)-cell enteropathy (NKCE) is recognized as a benign NK-cell lymphoproliferative disease. Due to its histological similarity to NK/T cell lymphoma, it is easy to misdiagnose, leading to unnecessary chemotherapy and poor quality of life. This disease is typically observed in the small and large intestines in North America, whereas almost all cases in Japan occur locally in the stomach. Only 11 LyGa/NKCE cases involving both gastric and intestinal lesions have been reported, and there are few reports providing endoscopic images throughout the gastrointestinal tract. We report a case of LyGa/NKCE involving both the stomach and small and large intestines with detailed upper gastrointestinal endoscopy, colonoscopy, capsule endoscopy and pathology images. Its pathogenesis currently remains elusive, but most patients with LyGa/NKCE in Japan have Helicobacter pylori (H. pylori) infection. Our patient was also positive for H. pylori infection at disease onset, but after receiving eradication therapy, ulcerative lesions in both stomach and intestine regressed and no recurrence was observed. This case suggests a link between the pathogenesis of LyGa/NKCE and H. pylori infection.</p>

Lectin staining analysis of protein glycosylation in urothelial carcinomas

M Fujimoto, E Arai, N Nishiyama, A Maeshima, H Fujimoto, H Kitamura, ...

CANCER SCIENCE 113, 378-378 113 378 - 378 2022.02

ISSN 1347-9032

Integrated multi-omics analysis in pathological tissue specimens

Y Kanai

CANCER SCIENCE 113, 872-872 113 872 - 872 2022.02

Lead author, Last author, Corresponding author, ISSN 1347-9032

Epigenome analysis using pathological tissue specimens for personalized treatment and prevention

Y Kanai

CANCER SCIENCE 113 113 2022.02

Lead author, Last author, Corresponding author, ISSN 1347-9032

Discovery of actionable vulnerabilities in scirrhous-type gastric cancer

Y Tanaka, F Chiwaki, S Kojima, M Kawazu, M Komatsu, T Ueno, S Inoue, ...

CANCER SCIENCE 113 (WILEY) 113 2022.02

Accepted, ISSN 1347-9032

Carcinogenic risk estimation by DNA methylation quantification in patients with nonalcoholic steatohepatitis

J Kuramoto, E Arai, Y Tian, S Makiuchi, N Tsuda, H Ojima, Y Yamada, ...

CANCER SCIENCE 113 113 2022.02

Last author, ISSN 1347-9032

Alteration of protein glycosylation in renal carcinogenesis

Kitazume Yoshiko, Arai Eri, Matsuda Atsushi, Kakuda Shuichi, Ohara Kentaro, Maeshima Akiko, Kuno Atsushi, Yoshida Teruhiko, Kanai Yae

CANCER SCIENCE 113 1284 - 1284 2022.02

ISSN 1347-9032

Method for determining prognosis of endometrial cancer

Y Kanai, E Arai, D Aoki, N Susumu, W Yamagami, T Makabe

US Patent App. 17/298,863 2022

Quantification of DNA methylation for carcinogenic risk estimation in patients with non-alcoholic steatohepatitis

J Kuramoto, E Arai, M Fujimoto, Y Tian, Y Yamada, T Yotani, S Makiuchi, ...

Cancer Research 82 (12_Supplement), 5703-5703 (Clinical Epigenetics) 14 ( 1 ) 168 - 168 2022

ISSN 18687075

Background: In recent years, non-alcoholic steatohepatitis (NASH) has become the main cause of hepatocellular carcinoma (HCC). As a means of improving the treatment of NASH-related HCCs based on early detection, this study investigated the feasibility of carcinogenic risk estimation in patients with NASH. Results: Normal liver tissue (NLT), non-cancerous liver tissue showing histological findings compatible with non-alcoholic fatty liver from patients without HCC (NAFL-O), non-cancerous liver tissue showing NASH from patients without HCC (NASH-O), non-cancerous liver tissue showing non-alcoholic fatty liver from patients with HCC (NAFL-W), non-cancerous liver tissue showing NASH from patients with HCC (NASH-W) and NASH-related HCC were analyzed. An initial cohort of 171 tissue samples and a validation cohort of 55 tissue samples were used. Genome-wide DNA methylation screening using the Infinium HumanMethylation450 BeadChip and DNA methylation quantification using high-performance liquid chromatography (HPLC) with a newly developed anion-exchange column were performed. Based on the Infinium assay, 4050 CpG sites showed alterations of DNA methylation in NASH-W samples relative to NLT samples. Such alterations at the precancerous NASH stage were inherited by or strengthened in HCC samples. Receiver operating characteristic curve analysis identified 415 CpG sites discriminating NASH-W from NLT samples with area under the curve values of more than 0.95. Among them, we focused on 21 CpG sites showing more than 85% specificity, even for discrimination of NASH-W from NASH-O samples. The DNA methylation status of these 21 CpG sites was able to predict the coincidence of HCC independently from histopathological findings such as ballooning and fibrosis stage. The methylation status of 5 candidate marker CpG sites was assessed using a HPLC-based system, and for 3 of them sufficient sensitivity and specificity were successfully validated in the validation cohort. By combining these 3 CpG sites including the ZC3H3 gene, NAFL-W and NASH-W samples from which HCCs had already arisen were confirmed to show carcinogenic risk with 95% sensitivity in the validation cohort. Conclusions: After a further prospective validation study using a larger cohort, carcinogenic risk estimation in liver biopsy specimens of patients with NASH may become clinically applicable using this HPLC-based system for quantification of DNA methylation.

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Cancer-specific glycosylation regulated by epigenomic alteration in renal cell carcinoma

Y Kitazume, E Arai, A Matsuda, K Ohara, AM Maeshima, T Yoshida, ...

Cancer Research 82 (12_Supplement), 5702-5702 82 ( 12 ) 2022

ISSN 0008-5472

Potential DNA methylation biomarkers for the detection of clear cell renal cell carcinoma identified by a whole blood-based epigenome-wide association study

H Ohmomo, S Komaki, Y Sutoh, T Hachiya, K Ono, E Arai, H Fujimoto, ...

Epigenetics Communications 2 (1), 1-11 2022

Accepted

The oligodendroglial histological features are not independently predictive of patient prognosis in lower-grade gliomas

ES Pareira, M Shibuya, K Ohara, Y Nakagawa, T Kanazawa, ...

Brain tumor pathology, 1-9 (Brain Tumor Pathology) 39 ( 2 ) 79 - 87 2022

Accepted, ISSN 14337398

The relevance of oligodendroglial histological features to patient prognoses is controversial. 93 LrGGs resected for about 2 decades were re-assessed based on WHO2007 with special interest to pure oligodendroglial diagnosis (oligodendroglioma or anaplastic oligodendroglioma) and presence of CFO features. Those histological features, patients OS, and tumor chromosomal/genetic characteristics were correlated each other in each of the 3 IDH-1p/19q-based molecular groups. There was significant association between 1p19q status with the oligodendroglial histological diagnosis as well as presence of CFO in the entire cohort. The oligodendroglial diagnosis was associated with longer OS in IDHmut/codel group; however, this association was not significant in the multivariate analyses. In IDHmut/noncodel and IDH-wildtype groups, the oligodendroglial diagnosis was not associated with patient OS. Presence of CFO was not associated with patient OS in any molecular groups. Gain of 8q was associated with the oligodendroglial diagnosis in IDHmut/noncodel group. Neither the oligodendroglial diagnosis nor CFO was predictive for the methylation status of the MGMT gene in any molecular groups. The oligodendroglial histological features are not independently predictive of either patient prognosis or chemotherapeutic response in LrGGs, leaving the possibility of marginal favorable association only in IDHmut/codel tumors.

DNA methylation status of the SPHK1 and LTB genes underlies the clinicopathological diversity of non-alcoholic steatohepatitis-related hepatocellular carcinomas

N Tsuda, Y Tian, M Fujimoto, J Kuramoto, S Makiuchi, H Ojima, M Gotoh, ...

Journal of Cancer Research and Clinical Oncology, 1-17 (Journal of Cancer Research and Clinical Oncology) 149 ( 8 ) 5109 - 5125 2022

ISSN 01715216

Purpose: This study was performed to identify the DNA methylation profiles underlying the clinicopathological diversity of non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinomas (HCCs). Methods: Genome-wide DNA methylation analysis of 88 liver tissue samples was performed using the Infinium assay. Results: Principal component analysis revealed that distinct DNA methylation profiles differing from such profiles in normal control liver tissue had already been established in non-cancerous liver tissue showing NASH, which is considered to be a precancerous condition. Hierarchical clustering separated 26 NASH-related HCCs into Cluster I (n = 8) and Cluster II (n = 18). Such epigenetic clustering was significantly correlated with histopathological diversity, i.e. poorer tumor differentiation, tumor steatosis and development of a scirrhous HCC component. Significant differences in DNA methylation levels between the two clusters were accumulated in molecular pathways participating in cell adhesion and cytoskeletal remodeling, as well as cell proliferation and apoptosis. Among tumor-related genes characterizing Clusters I and II, differences in the levels of DNA methylation and mRNA expression for the SPHK1, INHBA, LTB and PDE3B genes were correlated with poorer tumor differentiation. 5-Aza-2′-deoxycytidine treatment of HCC cells revealed epigenetic regulation of the SPHK1 and LTB genes. Knockdown experiments showed that SPHK1 promotes cell proliferation, represses apoptosis and enhances migration, whereas LTB enhances migration of HCC cells. DNA hypomethylation resulting in increased expression of SPHK1 and LTB in poorly differentiated HCCs may underlie the aggressive phenotype of such HCCs. Conclusion: These data indicate that DNA methylation profiles may determine the clinicopathological heterogeneity of NASH-related HCCs via alterations of tumor-related gene expression.

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The Japanese Society of Pathology Practical Guidelines on the handling of pathological tissue samples for cancer genomic medicine.

Y Hatanaka, T Kuwata, E Morii, Y Kanai, H Ichikawa, T Kubo, ...

Pathology International (Pathology International) 71 ( 11 ) 725 - 740 2021.10

Joint Work, Accepted, ISSN 1320-5463

Clinical cancer genomic testing based on next-generation sequencing can help select genotype-matched therapy and provide diagnostic and prognostic information. Pathological tissue from malignant tumors obtained during routine practice are frequently used for genomic testing. This article is aimed to standardize the proper handling of pathological specimens in practice for genomic medicine based on the findings established in “Guidelines on the handling of pathological tissue samples for genomic medicine (in Japanese)” published by The Japanese Society of Pathology (JSP) in 2018. The two-part practical guidelines are based on empirical data analyses; Part 1 describes the standard preanalytic operating procedures for tissue collection, processing, and storage of formalin-fixed paraffin-embedded (FFPE) samples, while Part 2 describes the assessment and selection of FFPE samples appropriate for genomic testing, typically conducted by a pathologist. The guidelines recommend that FFPE sample blocks be used within 3 years from preparation, and the tumor content should be ≥30% (minimum 20%). The empirical data were obtained from clinical studies performed by the JSP in collaboration with leading Japanese cancer genome research projects. The Japanese Ministry of Health, Labour, and Welfare (MHLW) recommended to comply with the JSP practical guidelines in implementing cancer genomic testing under the national health insurance system in over 200 MHLW-designated core and cooperative cancer genome medicine hospitals in Japan.

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Molecular classification of gastric cancer predicts survival in patients undergoing radical gastrectomy based on project HOPE

K Furukawa, K Hatakeyama, M Terashima, T Nagashima, K Urakami, ...

Gastric Cancer, 1-11 (Gastric Cancer) 25 ( 1 ) 138 - 148 2021.09

Joint Work, Accepted, ISSN 1436-3291

Background: Gastric cancer (GC) has been classified based on molecular profiling like The Cancer Genome Atlas (TCGA) and Asian Cancer Research Group (ACRG), and attempts have been made to establish therapeutic strategies based on these classifications. However, it is difficult to predict the survival according to these classifications especially in radically resected patients. We aimed to establish a new molecular classification of GC which predicts the survival in patients undergoing radical gastrectomy. Methods: The present study included 499 Japanese patients with advanced GC undergoing radical (R0/R1) gastrectomy. Whole-exome sequencing, panel sequencing, and gene expression profiling were conducted (High-tech Omics-based Patient Evaluation [Project HOPE]). We classified patients according to TCGA and ACRG subtypes, and evaluated the clinicopathologic features and survival. Then, we attempted to classify patients according to their molecular profiles associated with biological features and survival (HOPE classification). Results: TCGA and ACRG classifications failed to predict the survival. In HOPE classification, hypermutated (HMT) tumors were selected first as a distinctive feature, and T-cell-inflamed expression signature-high (TCI) tumors were then extracted. Finally, the remaining tumors were divided by the epithelial-mesenchymal transition (EMT) expression signature. HOPE classification significantly predicted the disease-specific and overall survival (p < 0.001 and 0.020, respectively). HMT + TCI showed the best survival, while EMT-high showed the worst survival. The HOPE classification was successfully validated in the TCGA cohort. Conclusions: We established a new molecular classification of gastric cancer that predicts the survival in patients undergoing radical surgery.

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【病理医が知っておくべき法令や指針】Q&A 剖検

若狹朋子, 木ノ元直樹, 種井善一, 金井弥栄, 柴原純二

病理と臨床 ((株)文光堂) 39 ( 9 ) 904 - 908 2021.09

ISSN 0287-3745

Brain Pathology of 18 Supercentenarians Aged 110 And Over

Takao Masaki, Mihara Ban, Arai Yasumichi, Murakami Tomoyuki, Nose Soichiro, Hamaya Kazuo, Nishida Katsuya, Futamura Naonobu, Nakayama Yuji, Mizutani Masashi, Sano Terunori, Kanai Yae, Mimura Masaru, Okano Hideyuki, Hirose Nobuyoshi

JOURNAL OF NEUROPATHOLOGY AND EXPERIMENTAL NEUROLOGY (OXFORD UNIV PRESS INC) 80 ( 6 ) 594 - 594 2021.06

Joint Work, ISSN 0022-3069

Demarcated redness associated with increased vascular density/size: a useful marker of flat-type dysplasia in patients with ulcerative colitis.

Ikebata A, Shimoda M, Okabayashi K, Uraoka T, Maehata T, Sugimoto S, Mutaguchi M, Naganuma M, Kameyama K, Yahagi N, Kanai T, Kitagawa Y, Kanai Y, Iwao Y

Endoscopy international open 9 ( 4 ) E552 - E561 2021.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 2364-3722

Background and study aims Recent advances in endoscopic equipment and diagnostic techniques have improved the detection of dysplasia in the inflamed mucosa of patients with ulcerative colitis (UC). However, it remains difficult to endoscopically identify flat-type dysplasia which has been formerly recognized as invisible dysplasia. Patients and methods In this retrospective, single-center study, we endoscopically identified 10 cases of flat-type-predominant dysplasia by targeted biopsy among 38 intramucosal dysplasia lesions from patients with UC who underwent surgical or endoscopic resection from 2007 to 2017. Their endoscopic and histological features were examined, including color changes, intramucosal vascular density/size, and vascular endothelial growth factor (VEGF) expression. Results All flat-type-predominant dysplasias were endoscopically recognized as demarcated red-colored areas and histologically diagnosed as low- (LGDs) or high-grade dysplasias (HGDs). Immunohistochemical examination using resected specimens revealed that flat-type dysplasia was characterized by significantly increased CD34-positive vascular density (LGDs, 1.7-fold, P < 0.01; HGDs, 2.2-fold, P < 0.01) and size (LGDs, 1.03-fold, P < 0.01; HGDs, 1.11-fold, P < 0.01) in the mucosa, compared to adjacent non-neoplastic areas. Increased numbers of vessels were observed at the base of the mucosa in LGDs, whereas HGDs contained increased/enlarged vessels throughout the mucosa. Moreover, VEGF expression was elevated in all dysplastic epithelia. Conclusions Demarcated red-colored areas, histologically characterized by an increased vascular density/size in the mucosa, are an endoscopic sign of formerly invisible flat-type dysplasia in patients with UC and should be considered for targeted biopsy. Prospective studies focusing on the mucosal color change for their early detection would be desirable in the future.

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Alterations of protein glycosylation and DNA methylation in urothelial carcinoma

Fujimoto Mao, Arai Eri, Matsuda Atsushi, Maeshima Akiko, Fujimoto Hiroyuki, Kuno Atsushi, Yoshida Teruhiko, Kanai Yae

CANCER SCIENCE 112 670 - 670 2021.02

Joint Work, ISSN 1347-9032

Infiltration of TAMs is involved in tumor PD-L1 expression in early lung adenocarcinoma

Shima Toshiyuki, Shimoda Masayuki, Shigenobu Takao, Otsuka Takashi, Nishimura Tomoyasu, Emoto Katsura, Hayashi Yuichirou, Iwasaki Tatusrou, Abe Takayuki, Asamura Hisao, Kanai Yae

CANCER SCIENCE 112 397 - 397 2021.02

Joint Work, Accepted, ISSN 1347-9032

Epigenomic alterations during developmental stages of non-inflammatory hepatocellular carcinoma of unknown etiology

Makiuchi Satomi, Arai Eri, Kuramoto Junko, Tian Ying, Tsuda Noboru, Ojima Hidenori, Takahashi Yoriko, Hiraoka Nobuyoshi, Yoshida Teruhiko, Kanai Yae

CANCER SCIENCE 112 674 - 674 2021.02

Joint Work, ISSN 1347-9032

Epigenomic alterations and histological heterogeneity of non-alcoholic steatohepatitis-related hepatocellular carcinomas

Tsuda Noboru, Arai Eri, Kuramoto Junko, Tian Ying, Makiuchi Satomi, Ojima Hidenori, Takahashi Yoriko, Hiraoka Nobuyoshi, Yoshida Teruhiko, Kanai Yae

CANCER SCIENCE 112 671 - 671 2021.02

Joint Work, ISSN 1347-9032

腸に限局した原発性AL型アミロイドーシスの4例

中島真, 下田将之, 岩男泰, 川井田みほ, 亀山香織, 内木宏延, 金井弥栄

診断病理 ((一社)日本病理学会) 38 ( 1 ) 77 - 81 2021.01

Joint Work, Accepted, ISSN 1345-6431

消化管に限局した原発性アミロイドーシスは稀な疾患である。今回4例の腸限局性アミロイドーシスを経験した。いずれの症例も内視鏡的に大腸を中心とした多発性黄白色調粘膜下腫瘍様隆起として観察され、組織学的にはAL(λ)型アミロイド沈着を認めた。経過観察可能だった3例はいずれも生命予後良好で、進行は緩徐であった。また、非隆起部からアミロイド沈着初期像を検出するとともに、アミロイド沈着部にはCD34陽性細胞の軽度集簇が観察され、これらの所見はアミロイド沈着機序理解の新たな糸口となる可能性が示唆された。(著者抄録)

Multi-omic profiling of peritoneal metastases in gastric cancer identifies molecular subtypes and therapeutic vulnerabilities

Y Tanaka, F Chiwaki, S Kojima, M Kawazu, M Komatsu, T Ueno, S Inoue, ...

Nature Cancer, 1-16 (Nature Cancer) 2 ( 9 ) 962 - 977 2021

Joint Work

Peritoneal metastasis, a hallmark of incurable advanced gastric cancer (GC), presently has no curative therapy and its molecular features have not been examined extensively. Here we present a comprehensive multi-omic analysis of malignant ascitic fluid samples and their corresponding tumor cell lines from 98 patients, including whole-genome sequencing, RNA sequencing, DNA methylation and enhancer landscape. We identify a higher frequency of receptor tyrosine kinase and mitogen-activated protein kinase pathway alterations compared to primary GC; moreover, approximately half of the gene alterations are potentially treatable with targeted therapy. Our analyses also stratify ascites-disseminated GC into two distinct molecular subtypes: one displaying active super enhancers (SEs) at the ELF3, KLF5 and EHF loci, and a second subtype bearing transforming growth factor-β (TGF-β) pathway activation through SMAD3 SE activation and high expression of transcriptional enhancer factor TEF-1 (TEAD1). In the TGF-β subtype, inhibition of the TEAD pathway circumvents therapy resistance, suggesting a potential molecular-guided therapeutic strategy for this subtype of intractable GC.

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Clinicopathological impacts of DNA methylation alterations on pancreatic ductal adenocarcinoma: prediction of early recurrence based on genome-wide DNA methylation profiling

Endo Y, Fujimoto M, Ito N, Takahashi Y, Kitago M, Gotoh M, Hiraoka N, Yoshida T, Kitagawa Y, Kanai Y, Arai E

Journal of Cancer Research and Clinical Oncology (Journal of Cancer Research and Clinical Oncology) 147 ( 5 ) 1341 - 1354 2021

Joint Work, Accepted, ISSN 01715216

© 2021, The Author(s). Purpose: The present study was conducted to clarify the clinicopathological impacts of DNA methylation alterations on pancreatic ductal adenocarcinoma (PDAC). Methods: Genome-wide DNA methylation screening was performed using the Infinium HumanMethylation450 BeadChip, and DNA methylation quantification was verified using pyrosequencing. We analyzed fresh-frozen tissues from an initial cohort (17 samples of normal control pancreatic tissue [C] from 17 patients without PDAC, and 34 samples of non-cancerous pancreatic tissue [N] and 82 samples of cancerous tissue [T] both obtained from 82 PDAC patients) and formalin-fixed paraffin-embedded T samples from 34 patients in a validation cohort. Results: The DNA methylation profiles of N samples tended to differ from those of C samples, and 91,907 probes showed significant differences in DNA methylation levels between C and T samples. Epigenetic clustering of T samples was significantly correlated with a larger tumor diameter and early recurrence (ER), defined as relapse within 6 months after surgery. Three marker CpG sites, applicable to formalin-fixed paraffin-embedded surgically resected materials regardless of their tumor cell content, were identified for prediction of ER. The sensitivity and specificity for detection of patients belonging to the ER group using a panel combining these three marker CpG sites, including a CpG site in the CDK14 gene, were 81.8% and 71.7% and 88.9% and 70.4% in the initial and validation cohorts, respectively. Conclusion: These findings indicate that DNA methylation alterations may have a clinicopathological impact on PDAC. Application of our criteria will ultimately allow prediction of ER after surgery to improve the outcome of PDAC patients.

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Evaluation of clinical formalin‐fixed paraffin‐embedded tissue quality for targeted‐bisulfite sequencing

H Ohmomo, S Komaki, K Ono, Y Sutoh, T Hachiya, E Arai, H Fujimoto, ...

Pathology international 71 ( 2 ) 135 - 140 2021

Accepted

DNA hypermethylation of the ZNF132 gene participates in the clinicopathological aggressiveness of ‘pan-negative’-type lung adenocarcinomas

K Hamada, Y Tian, M Fujimoto, Y Takahashi, T Kohno, K Tsuta, ...

Carcinogenesis 42 (2), 169-179 2021

Accepted

CD163-positive cancer cells are a predictor of a worse clinical course in lung adenocarcinoma

Matsubara E, Komohara Y, Shinchi Y, Mito R, Fujiwara Y, Ikeda K, Shima T, Shimoda M, Kanai Y, Sakagami T, Suzuki M

Pathology International (Pathology International) 71 ( 10 ) 666 - 673 2021

Joint Work, Accepted, ISSN 13205463

CD163 is one of the scavenger receptors expressed on macrophages. However, several immunohistochemical studies have demonstrated that CD163 is also detected on cancer cells, and is associated with a poor prognosis. In the present study, we detected CD163 staining on cancer cells in lung adenocarcinoma and squamous cell carcinoma (SCC), and investigated the relationship between CD163 on cancer cells and the clinical prognosis. CD163 staining was seen in 128 of 342 adenocarcinoma cases and 35 of 103 SCC cases. Among the lung adenocarcinoma cases, the progression-free survival and overall survival were significantly shorter in the CD163 high group than the CD163 low group. A similar trend was observed among the SCC cases, but the difference was not statistically significant. Additionally, a higher number of macrophages was detected in areas with CD163-positive cancer cells when compared to areas with CD163-negative cancer cells. In summary, we found that CD163-positive cancer cells are a predictor of a worse clinical course in lung adenocarcinoma and SCC.

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Method for determining risk of urothelial carcinoma

Y Kanai, E Arai

US Patent App. 16/970,583 2021

Correaltions between genome-wide DNA methylation profiles and genomic driver aberrations during multistage lung adenocaricinogenesis

K Hamada, Y Tian, M Fujimoto, T Yoriko, T Kohno, K Tsuta, S Watanabe, ...

Cancer Research 81 (13 Supplement), 2094-2094 81 ( 13 ) 2021

Joint Work, ISSN 0008-5472

Prognostication of early-onset endometrioid endometrial cancer based on genome-wide DNA methylation profiles

T Hirano, E Arai, M Fujimoto, Y Nakayama, Y Tian, N Ito, T Makabe, ...

Journal of Gynecologic Oncology 33 (Journal of Gynecologic Oncology) 33 ( 6 ) e74 2021

ISSN 2005-0380

Objective: The aim of this study was to establish criteria that would indicate whether fertility preservation therapy would likely be safe for patients aged 40 years or less with endometrioid endometrial cancer based on their DNA methylation profile. Methods: Forty-nine fresh-frozen tissue samples from patients with endometrial cancer from an initial cohort and 31 formalin-fixed paraffin-embedded tissue samples from a second cohort were subjected to genome-wide DNA methylation analysis using the Infinium MethylationEPIC BeadChip. Results: Epigenomic clustering of early-onset endometrial cancer was correlated with the widely used recurrence risk classification. Genes showing differences in DNA methylation levels between the low-recurrence-risk category and intermediate-and high-risk categories were accumulated in pathways related to fibroblast growth factor and nuclear factor-κB signaling. DNA hypomethylation and overexpression of ZBTB38 were frequently observed in the low-risk category. Eight hundred thirty-one marker CpG probes showed area under the curve values of >0.7 on the receiver operating characteristic curve for discrimination of patients belonging to the low-risk category. By combining marker CpG sites, seven panels for placing patients into the low-risk category with 91.3% or more sensitivity and specificity in both the initial and second cohorts were established. Conclusions: DNA methylation diagnostics criteria using up to 6 of 8 CpG sites for LPP, FOXO1, RNF4, EXOC6B, CCPG1, RREB1 and ZBTB38 may be applicable to recurrence risk estimation for patients aged 40 years or less with endometrial cancer, regardless of tumor cell content, even if formalin-fixed paraffin-embedded biopsy or curettage materials are used.

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超百寿者エピゲノム解析による健康長寿規定要因の解明

金井弥栄

上原記念生命科学財団研究報告集 ((公財)上原記念生命科学財団) 34 1 - 5 2020.12

Single Work

DNA hypermethylation of the ZNF132 gene participates in the clinicopathological aggressiveness of "pan-negative"-type lung adenocarcinomas.

Hamada K, Tian Y, Fujimoto M, Takahashi Y, Kohno T, Tsuta K, Watanabe SI, Yoshida T, Asamura H, Kanai Y, Arai E

Carcinogenesis (Carcinogenesis) 42 ( 2 ) 169 - 179 2020.11

Joint Work, Accepted, ISSN 0143-3334

© The Author(s) 2020. Published by Oxford University Press. Although some previous studies have examined epigenomic alterations in lung adenocarcinomas, correlations between epigenomic events and genomic driver mutations have not been fully elucidated. Single-CpG resolution genome-wide DNA methylation analysis with the Infinium HumanMethylation27 BeadChip was performed using 162 paired samples of adjacent normal lung tissue (N) and the corresponding tumorous tissue (T) from patients with lung adenocarcinomas. Correlations between DNA methylation data on the one hand and clinicopathological parameters and genomic driver mutations, i.e. mutations of EGFR, KRAS, BRAF and HER2 and fusions involving ALK, RET and ROS1, were examined. DNA methylation levels in 12 629 probes from N samples were significantly correlated with recurrence-free survival. Principal component analysis revealed that distinct DNA methylation profiles at the precancerous N stage tended not to induce specific genomic driver aberrations. Most of the genes showing significant DNA methylation alterations during transition from N to T were shared by two or more driver aberration groups. After small interfering RNA knockdown of ZNF132, which showed DNA hypermethylation only in the pan-negative group and was correlated with vascular invasion, the proliferation, apoptosis and migration of cancer cell lines were examined. ZNF132 knockdown led to increased cell migration ability, rather than increased cell growth or reduced apoptosis. We concluded that DNA hypermethylation of the ZNF132 gene participates in the clinicopathological aggressiveness of 'pan-negative' lung adenocarcinomas. In addition, DNA methylation alterations at the precancerous stage may determine tumor aggressiveness, and such alterations that accumulate after driver mutation may additionally modify clinicopathological features through alterations of gene expression.

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Stromal metalloproteinases: Crucial contributors to the tumor microenvironment.

Shimoda M, Ohtsuka T, Okada Y, Kanai Y

Pathology international (Pathology International) 71 ( 1 ) 1 - 14 2020.10

Joint Work, Last author, Accepted, ISSN 1320-5463

© 2020 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd Proteolytic balance is crucial for the maintenance of tissue homeostasis. In cancer, dysregulated proteolysis is involved in unregulated tissue remodeling and inflammation, leading to the promotion of tumor growth, local invasion, and metastasis. Metalloproteinases, which were first identified as collagen cleaving enzymes, have been shown to extensively degrade extracellular matrix proteins or selectively release cell surface-bound cytokines, growth factors, or their receptors, thereby impacting extracellular matrix integrity, immune cell recruitment and tissue turnover. Although tumor cells produce various metalloproteinases, the major source is thought to be stromal cells infiltrating the tumor. Different types of stromal cells express specific sets of metalloproteinases and their inhibitors, which specifically alter the milieu within the tumor. In this review, recent findings and knowledge regarding metalloproteinases derived from stromal cells during the creation of the tumor microenvironment are described and their contribution to the tumor progression and metastasis discussed.

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尿路上皮がんにおける糖鎖発現異常ならびに糖転移酵素のDNAメチル化異常

藤本真央, 新井恵吏, 松田厚志, 前島亜希子, 藤元博行, 久野敦, 吉田輝彦, 金井弥栄

日本癌学会総会記事 ((一社)日本癌学会) 79回 PE9 - 1 2020.10

ISSN 0546-0476

Methylation pattern of urinary DNA as a marker of kidney function decline in diabetes

Marumo T, Hoshino J, Kawarazaki W, Nishimoto M, Ayuzawa N, Hirohama D, Yamanouchi M, Ubara Y, Okaneya T, Fujii T, Yuki K, Atsumi Y, Sato A, Arai E, Kanai Y, Shimosawa T, Fujita T

BMJ open diabetes research & care (BMJ open diabetes research & care) 8 ( 1 ) e001501 - e001501 2020.09

Research paper (scientific journal), Joint Work, Accepted

© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. INTRODUCTION: Renal tubular injury contributes to the decline in kidney function in patients with diabetes. Cell type-specific DNA methylation patterns have been used to calculate proportions of particular cell types. In this study, we developed a method to detect renal tubular injury in patients with diabetes by detecting exfoliated tubular cells shed into the urine based on tubular cell-specific DNA methylation patterns. RESEARCH DESIGN AND METHODS: We identified DNA methylation patterns specific for human renal proximal tubular cells through compartment-specific methylome analysis. We next determined the methylation levels of proximal tubule-specific loci in urine sediment of patients with diabetes and analyzed correlation with clinical variables. RESULTS: We identified genomic loci in SMTNL2 and G6PC to be selectively unmethylated in human proximal tubular cells. The methylation levels of SMTNL2 and G6PC in urine sediment, deemed to reflect the proportion of exfoliated proximal tubular cells due to injury, correlated well with each other. Methylation levels of SMTNL2 in urine sediment significantly correlated with the annual decline in estimated glomerular filtration rate. Moreover, addition of urinary SMTNL2 methylation to a model containing known risk factors significantly improved discrimination of patients with diabetes with faster estimated glomerular filtration rate decline. CONCLUSIONS: This study demonstrates that patients with diabetes with continual loss in kidney function may be stratified by a specific DNA methylation signature through epigenetic urinalysis and provides further evidence at the level of exfoliated cells in the urine that injury of proximal tubular cells may contribute to pathogenesis of diabetic kidney disease.

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Simple vs. comprehensive prediction models of homologous recombination deficiency based on mutational and clinical features in three independent breast cancer datasets

Tanioka Maki, Watanabe Tomoko, Honda Takayuki, Totsuka Hirohiko, Arai Eri, Kanai Yae, Shiraishi Kouya, Tamura Kenji, Kohno Takashi

CANCER RESEARCH 80 ( 16 ) 2020.08

Joint Work, Accepted, ISSN 0008-5472

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Cooperative participation of epigenomic and genomic alterations in the clinicopathological diversity of gastric adenocarcinomas: significance of cell adhesion and epithelial-mesenchymal transition-related signaling pathways.

Yang M, Arai E, Takahashi Y, Totsuka H, Chiku S, Taniguchi H, Katai H, Sakamoto H, Yoshida T, Kanai Y

Carcinogenesis (Carcinogenesis) 41 ( 11 ) 1473 - 1484 2020.07

Joint Work, Last author, Accepted, ISSN 0143-3334

© The Author(s) 2020. Published by Oxford University Press. The present study was conducted to clarify the cooperative significance of epigenomic and genomic abnormalities during gastric carcinogenesis. Using 21 samples of normal control gastric mucosa (C), 109 samples of non-cancerous gastric mucosa (N) and 105 samples of cancerous tissue (T) from 109 patients with primary gastric adenocarcinomas, genome-wide DNA methylation analysis was performed using Infinium assay. Among these samples, 66 paired N and corresponding T samples were subjected to whole-exome and single nucleotide polymorphism array analyses. As had been shown in our previous study, 109 patients were clustered clinicopathologically into least aggressive Cluster A (n = 20), most aggressive Cluster B1 (n = 20) and Cluster B2 (n = 69). Most DNA methylation alterations in each cluster had already occurred even in N samples compared with C samples, and DNA methylation alterations at the precancerous N stage were inherited by the established cancers themselves. Recurrent single nucleotide variants and insertions/deletions resulting in functional disruption of the proteins encoded by the ABCA10, BNC2, CDH1, CTNNB1, SMAD4 and VAV2 genes were specific to Cluster B1, whereas those of the APC, EGFR, ERBB2, ERBB3, MLH1 and MUC6 genes were specific to Cluster A. MetaCore pathway analysis revealed that the epigenomically affected TWIST1 gene and genomically affected CDH1, CTNNB1, MMP9, TLN2, ROCK1 and SMAD4 genes were accumulated in signaling pathways related to cell adhesion, cytoskeleton remodeling and epithelial-mesenchymal transition in Cluster B1. These data indicate that epigenomic alterations at the precancerous stage are important in gastric carcinogenesis and that epigenomic and genomic alterations cooperatively underlie the aggressiveness of gastric adenocarcinomas.

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Prophylactic therapy with human amniotic fluid stem cells improved survival in a rat model of lipopolysaccharide-induced neonatal sepsis through immunomodulation via aggregates with peritoneal macrophages.

Sato Y, Ochiai D, Abe Y, Masuda H, Fukutake M, Ikenoue S, Kasuga Y, Shimoda M, Kanai Y, Tanaka M

Stem cell research & therapy (Stem Cell Research and Therapy) 11 ( 1 ) 300 - 300 2020.07

Research paper (scientific journal), Joint Work, Accepted

BACKGROUND: Despite recent advances in neonatal care, sepsis remains a leading cause of mortality in neonates. Mesenchymal stem cells derived from various tissues, such as bone marrow, umbilical cord, and adipose tissue, have beneficial effects on adult sepsis. Although human amniotic fluid stem cells (hAFSCs) have mesenchymal stem cell properties, the efficacy of hAFSCs on neonatal sepsis is yet to be elucidated. This study aimed to investigate the therapeutic potential of hAFSCs on neonatal sepsis using a rat model of lipopolysaccharide (LPS)-induced sepsis. METHODS: hAFSCs were isolated as CD117-positive cells from human amniotic fluid. Three-day-old rat pups were intraperitoneally treated with LPS to mimic neonatal sepsis. hAFSCs were administered either 3 h before or at 0, 3, or 24 h after LPS exposure. Serum inflammatory cytokine levels, gene expression profiles from spleens, and multiple organ damage were analyzed. hAFSC localization was determined in vivo. In vitro LPS stimulation tests were performed using neonatal rat peritoneal macrophages co-cultured with hAFSCs in a cell-cell contact-dependent/independent manner. Immunoregulation in the spleen was determined using a DNA microarray analysis. RESULTS: Prophylactic therapy with hAFSCs improved survival in the LPS-treated rats while the hAFSCs transplantation after LPS exposure did not elicit a therapeutic response. Therefore, hAFSC pretreatment was used for all subsequent studies. Inflammatory cytokine levels were elevated after LPS injection, which was attenuated by hAFSC pretreatment. Subsequently, inflammation-induced damages in the brain, lungs, and liver were ameliorated. hAFSCs aggregated with peritoneal macrophages and/or transiently accumulated in the liver, mesentery, and peritoneum. Paracrine factors released by hAFSCs induced M1-M2 macrophage polarization in a cell-cell contact-independent manner. Direct contact between hAFSCs and peritoneal macrophages further enhanced the polarization. Microarray analysis of the spleen showed that hAFSC pretreatment reduced the expression of genes involved in apoptosis and inflammation and subsequently suppressed toll-like receptor 4 signaling pathways. CONCLUSIONS: Prophylactic therapy with hAFSCs improved survival in a rat model of LPS-induced neonatal sepsis. These effects might be mediated by a phenotypic switch from M1 to M2 in peritoneal macrophages, triggered by hAFSCs in a cell-cell contact-dependent/independent manner and the subsequent immunomodulation of the spleen.

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Aberrant DNA methylation results in altered gene expression in non-alcoholic steatohepatitis-related hepatocellular carcinomas.

Tian Y, Arai E, Makiuchi S, Tsuda N, Kuramoto J, Ohara K, Takahashi Y, Ito N, Ojima H, Hiraoka N, Gotoh M, Yoshida T, Kanai Y

Journal of cancer research and clinical oncology (Journal of Cancer Research and Clinical Oncology) 146 ( 10 ) 2461 - 2477 2020.07

Joint Work, Last author, Accepted, ISSN 0171-5216

© 2020, The Author(s). Purpose: The aim of this study was to investigate DNA methylation alterations in non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinomas (HCCs). Methods: Genome-wide DNA methylation analysis was performed using the Infinium Human Methylation 450 K BeadChip, and levels of mRNA expression were analyzed by quantitative reverse transcription-PCR. Results: Compared to 36 samples of normal control liver tissue (C), DNA methylation alterations were observed on 19,281 probes in 22 samples of cancerous tissue (T) obtained from patients showing histological features compatible with NASH in their non-cancerous liver tissue (N). Among those probes, 1396 were located within CpG islands or their shores and shelves, designed around the transcription start sites of 726 genes. In representative genes, such as DCAF4L2, CKLF, TRIM4, PRC1, UBE2C and TUBA1B, both DNA hypomethylation and mRNA overexpression were observed in T samples relative to C samples, and the levels of DNA methylation and mRNA expression were inversely correlated with each other. DNA hypomethylation occurred even in N samples at the precancerous NASH stage, and this was inherited by or further strengthened in T samples. DNA hypomethylation of DCAF4L2, CKLF and UBE2C was observed in both NASH-related and viral hepatitis-related HCCs, whereas that of TRIM4, PRC1 and TUBA1B occurred in a NASH-related HCC-specific manner. DNA hypomethylation and/or mRNA overexpression of these genes was frequently associated with the necroinflammatory grade of NASH and was correlated with poorer tumor differentiation. Conclusion: DNA methylation alterations may occur under the necroinflammatory conditions characteristic of NASH and participate in NASH-related hepatocarcinogenesis through aberrant expression of tumor-related genes.

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Establishment of diagnostic criteria for upper urinary tract urothelial carcinoma based on genome-wide DNA methylation analysis.

Fujimoto M, Arai E, Tsumura K, Yotani T, Yamada Y, Takahashi Y, Maeshima AM, Fujimoto H, Yoshida T, Kanai Y

Epigenetics (Epigenetics) 15 ( 12 ) 1 - 13 2020.06

Joint Work, Last author, Accepted, ISSN 1559-2294

© 2020, © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. The aim of this study was to develop a less invasive and accurate diagnostic system for upper urinary tract urothelial carcinoma (UTUC) based on genome-wide DNA methylation profiling. Genome-wide DNA methylation screening was performed using the Infinium HumanMethylation450 BeadChip, and DNA methylation quantification was verified using pyrosequencing. We analysed 26 samples of normal control urothelial tissue (C), an initial cohort of 62 samples (31 samples of non-cancerous urothelium [N] from UTUC patients and 31 samples of the corresponding UTUCs), a validation cohort of 82 samples (41 N and 41 UTUC samples), and 14 samples of urinary bladder urothelial carcinoma (BUC). In the initial cohort, we identified 2,448 CpG sites showing significant differences in DNA methylation levels between both C and UTUC and N and UTUC, but not showing differences between C and N. Among these CpG sites, 10 were located within CpG islands or their shores and shelves included in genomic domains where DNA methylation levels are stably controlled, allowing discrimination of UTUC even from BUC. Receiver operating characteristic curve analysis for discrimination of UTUC from N in these 10 CpG and neighbouring sites (37 diagnostic panels in total) yielded area under the curve values of 0.959–1.000, with a sensitivity and specificity of 86.6–100% and 93.5–100%, respectively. The diagnostic impact was successfully confirmed in the validation cohort. Our criteria were useful for diagnosis of UTUC, regardless of its clinicopathological features. Application of our criteria to voided urine samples will ultimately allow non-invasive DNA methylation diagnosis of UTUC.

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増刊号特集泌尿器科診療の最新スタンダード-平成の常識は令和の非常識腫瘍部位別腎細胞癌:病理診断

三上修治, 新井恵吏, 金井弥栄

臨床泌尿器科 (株式会社医学書院) 74 ( 4 ) 10 - 15 2020.04

ISSN 0385-2393

Infiltration of tumor-associated macrophages is involved in tumor programmed death-ligand 1 expression in early lung adenocarcinoma

Shima T, Shimoda M, Shigenobu T, Ohtsuka T, Nishimura T, Emoto K, Hayashi Y, Iwasaki T, Abe T, Asamura H, Kanai Y

Cancer Science (Cancer Science) 111 ( 2 ) 727 - 738 2020.02

Research paper (scientific journal), Joint Work, Last author, Accepted, ISSN 13479032

© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. Programmed death-ligand 1 (PD-L1) is an immune modulator that promotes immunosuppression by binding to programmed death-1 of T-lymphocytes. Although tumor cell PD-L1 expression has been shown to be associated with the clinical response to anti–PD-L1 antibodies, its concise regulatory mechanisms remain elusive. In this study, we evaluated the associations of tumor PD-L1 expression and immune cell infiltrating patterns in 146 cases of early lung adenocarcinoma (AC) to investigate the possible extrinsic regulation of tumor PD-L1 by immune cells. Using immunohistochemistry, cell surface PD-L1 expression in tumor cells was observed in 18.5% of stage 0-IA lung AC patients. Tumor PD-L1 positivity was significantly associated with stromal invasion, which was accompanied by increased tumor-associated macrophages (TAM), CD8+ cytotoxic T cells and FoxP3+ regulatory T cells. Among these immune cells, TAM and CD8+ T cells significantly accumulated in PD-L1-positive carcinoma cell areas, which showed a tumor cell nest-infiltrating pattern. Although CD8+ T cells are known to induce tumor PD-L1 expression via interferon-ɣ production, the increased TAM within tumors were also associated with tumor cell PD-L1 positivity, independently of CD8+ T cell infiltration. Our in vitro experiments revealed that PD-L1 expression in lung cancer cell lines was significantly upregulated by co–culture with M2-differentiated macrophages; expression of PD-L1 was reduced to baseline levels following treatment with a transforming growth factor-β inhibitor. These results demonstrated that tumor-infiltrating TAM are extrinsic regulators of tumor PD-L1 expression, indicating that combination therapy targeting both tumor PD-L1 and stromal TAM might be a possible strategy for effective treatment of lung cancer.

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Characteristics of leiomyosarcoma: Induction of hematogenous metastasis by isolated uterine mesenchymal tumor stem-like cells

Hayashi T, Sano K, Ichimura T, Kanai Y, Zharhary D, Aburatani H, Yaegashi N, Konishi I

Anticancer Research (Anticancer Research) 40 ( 3 ) 1255 - 1265 2020

Research paper (scientific journal), Joint Work, Accepted, ISSN 02507005

© 2020 International Institute of Anticancer Research. All rights reserved. Background/Aim: Uterine leiomyosarcoma (Ut- LMS) is a refractory tumor that repeatedly recurs with hematogenous metastasis, which may be due to the presence of drug-resistant tumor stem cells. Its treatment is limited to surgical procedures. We previously reported that Ut-LMS spontaneously developed in mice deficient in the proteasome component low-molecular mass polypeptide 2 (LMP2). We showed that LMP2 expression was significantly attenuated specifically in human Ut-LMS. The aim of this study was to investigate the role of LMP2 in hematogenous metastasis using xenograft models with tumor stem-like cells. Materials and Methods: We isolated tumor stem-like cells from LMP2- negative primary human Ut-LMS cells established from a human Ut-LMS tissue using the side population (SP) procedure. These cells were used to develop xenograft models with tumor stem-like cells. Results: Human Ut-LMS stem-like cells showed stronger hematogenous metastatic potential than normal Ut-LMS cells. Tumor stem-like cells also had the potential to differentiate into vascular endothelial cells through VEGF-A signaling. Conclusion: These results reflect frequent hematogenous metastasis by human Ut-LMS in clinical settings, and may lead to the development of treatments that inhibit hematogenous metastasis in Ut-LMS.

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Simple prediction model for homologous recombination deficiency in breast cancers in adolescents and young adults

Watanabe T, Honda T, Totsuka H, Yoshida M, Tanioka M, Shiraishi K, Shimada Y, Arai E, Ushiama M, Tamura K, Yoshida T, Kanai Y, Kohno T

Breast Cancer Research and Treatment (Breast Cancer Research and Treatment) 182 ( 2 ) 491 - 502 2020

Research paper (scientific journal), Joint Work, Accepted, ISSN 01676806

© 2020, Springer Science+Business Media, LLC, part of Springer Nature. Purpose: Homologous recombination deficiency (HRD), which influences the efficacy of PARP inhibitor- and platinum agent-based therapies, is a prevalent phenotype of breast cancer in adolescents and young adults (AYAs; 15–39 years old). However, HRD score, indicating HRD status, is not routinely assessed in the breast oncology clinic, particularly in patients without germline BRCA1/2 mutations. Hence, we sought to develop a model for determining HRD status based on genetic and clinicopathological factors. Methods: Subjects were our own cohort of 46 Japanese AYA breast cancer patients and two existing breast cancer cohorts of US and European patients. Models for prediction of the HRD-high phenotype, defined as HRD score ≥ 42, were constructed by logistic regression analysis, using as explanatory variables genetic and clinicopathological factors assessable in the clinical setting. Results: In all three cohorts, the HRD-high phenotype was associated with germline BRCA1/2 mutation, somatic TP53 mutation, triple-negative subtype, and higher tumor grade. A model based on these four factors, developed using the US cohort, was validated in the Japanese and European AYA cases: area under the receiver operating characteristic curve [AUC] was 0.90 and 0.96, respectively. A model based on three factors excluding germline BRCA1/2 mutation also yielded high-predictive power in cases from these two cohorts without germline BRCA1/2 mutations: AUC was 0.92 and 0.90, respectively. Conclusions: The HRD-high phenotype of AYA breast cancer patients can be deduced from genomic and pathological factors that are routinely examined in the oncology clinic, irrespective of germline BRCA1/2 mutations.

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Prophylactic therapy with human amniotic fluid stem cells improved survival in a rat model of lipopolysaccharide-induced neonatal sepsis through immunomodulation via aggregates …

Y Sato, D Ochiai, Y Abe, H Masuda, M Fukutake, S Ikenoue, Y Kasuga, ...

Stem cell research & therapy 11 (1), 1-14 2020

Method for prognosis of renal cell carcinoma

Y Kanai, E Arai, Y Tian

US Patent App. 16/708,879 2020

Method for prognosis of renal cell carcinoma

Y Kanai, E Arai, Y Tian

US Patent App. 16/708,879 2020

Method for prognosis of renal cell carcinoma

Y Kanai, E Arai, Y Tian

US Patent App. 16/708,879 2020

Method for prognosis of renal cell carcinoma

Y Kanai, E Arai, Y Tian

US Patent App. 16/708,879 2020

Method for evaluating risk of hepatocellular carcinoma

Y Kanai, E Arai, T Yotani, E Sunamura

US Patent App. 16/637,076 2020

Method for evaluating risk of hepatocellular carcinoma

Y Kanai, E Arai, T Yotani, E Sunamura

US Patent App. 16/637,076 2020

Method for evaluating risk of hepatocellular carcinoma

Y Kanai, E Arai, T Yotani, E Sunamura

US Patent App. 16/637,076 2020

Infiltration of tumor‐associated macrophages is involved in tumor programmed death‐ligand 1 expression in early lung adenocarcinoma

T Shima, M Shimoda, T Shigenobu, T Ohtsuka, T Nishimura, K Emoto, ...

Cancer science 111 ( 2 ) 727 2020

Last author

Cooperative participation of epigenomic and genomic alterations in the clinicopathological diversity of gastric adenocarcinomas: significance of cell adhesion and epithelial …

M Yang, E Arai, Y Takahashi, H Totsuka, S Chiku, H Taniguchi, H Katai, ...

Carcinogenesis 41 (11), 1473-1484 2020

Cooperative participation of epigenomic and genomic alterations in the clinicopathological diversity of gastric adenocarcinomas: significance of cell adhesion and epithelial …

M Yang, E Arai, Y Takahashi, H Totsuka, S Chiku, H Taniguchi, H Katai, ...

Carcinogenesis 41 (11), 1473-1484 2020

Candidate molecules as diagnostic biomarker for human uterine mesenchymal tumors

T Hayashi, K Sano, T Ichimura, G Gur, P Yaish, D Zharhary, Y Kanai, ...

Annals of Cytology and Pathology 5 (1), 054-057 2020

腫瘍部位別腎細胞癌: 病理診断

S MIKAMI, ERI ARAI, YAE KANAI

臨床泌尿器科 74 (4), 10-15 2020

Profiling of Target Molecules for Immunotherapy in Mesenchymal Tumors.

Hayashi T, Ichimura T, Sano K, Tonegawa S, Kanai Y, Zharhary D, Aburatani H, Yaegashi N, Konishi I

Journal of clinical medicine research 11 ( 8 ) 609 - 613 2019.08

Joint Work, Accepted, ISSN 1918-3003

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Genome-wide DNA methylation profile of early-onset endometrial cancer: Its correlation with genetic aberrations and comparison with late-onset endometrial cancer

Makabe T., Arai E., Hirano T., Ito N., Fukamachi Y., Takahashi Y., Hirasawa A., Yamagami W., Susumu N., Aoki D., Kanai Y.

Carcinogenesis (Carcinogenesis) 40 ( 5 ) 611 - 623 2019.07

Research paper (scientific journal), Joint Work, Last author, Accepted, ISSN 01433334

© 2019 The Author(s) 2019. Published by Oxford University Press. The present study was performed to clarify the significance of DNA methylation alterations during endometrial carcinogenesis. Genome-wide DNA methylation analysis and targeted sequencing of tumor-related genes were performed using the Infinium MethylationEPIC BeadChip and the Ion AmpliSeq Cancer Hotspot Panel v2, respectively, for 31 samples of normal control endometrial tissue from patients without endometrial cancer and 81 samples of endometrial cancer tissue. Principal component analysis revealed that tumor samples had a DNA methylation profile distinct from that of control samples. Gene Ontology enrichment analysis revealed significant differences of DNA methylation at 1034 CpG sites between early-onset endometrioid endometrial cancer (EE) tissue (patients aged ≤40 years) and late-onset endometrioid endometrial cancer (LE) tissue, which were accumulated among 'transcriptional factors'. Mutations of the CTNNB1 gene or DNA methylation alterations of genes participating in Wnt signaling were frequent in EEs, whereas genetic and epigenetic alterations of fibroblast growth factor signaling genes were observed in LEs

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Integrated analysis of genetic and epigenetic alterations in gastric carcinomas

Yang Menghan, Arai Eri, Sakamoto Hiromi, Totsuka Hirohiko, Taniguchi Hirokazu, Katai Hitoshi, Yoshida Teruhiko, Kanai Yae

CANCER RESEARCH 79 ( 13 ) 2019.07

Last author, ISSN 0008-5472

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Establishment of permutation for cancer risk estimation in the urothelium based on genome-wide DNA methylation analysis.

Tsumura K, Arai E, Tian Y, Shibuya A, Nishihara H, Yotani T, Yamada Y, Takahashi Y, Maeshima AM, Fujimoto H, Nakagawa T, Kume H, Homma Y, Yoshida T, Kanai Y.

Carcinogenesis (Carcinogenesis) 40 ( 11 ) 1308 - 1319 2019.06

Research paper (scientific journal), Joint Work, Last author, Accepted, ISSN 0143-3334

The aim of this study was to establish permutation for cancer risk estimation in the urothelium. Twenty-six samples of normal control urothelium obtained from patients without urothelial carcinomas (C), 47 samples of non-cancerous urothelium without noticeable morphological changes obtained from patients with urothelial carcinomas (N), and 46 samples of the corresponding cancerous tissue (T) in the learning cohort and 64 N samples in the validation cohort, i.e. 183 tissue samples in total, were analyzed. Genome-wide DNA methylation analysis was performed using the Infinium HumanMethylation 450K BeadChip, and DNA methylation levels were verified using pyrosequencing and MassARRAY. Amplicon sequencing was performed using the GeneRead DNAseq Targeted Panels V2. Although N samples rarely showed genetic mutations or copy number alterations, they showed DNA methylation alterations at 2502 CpG sites compared to C samples, and such alterations were inherited by or strengthened in T samples, indicating that DNA methylation alterations may participate in field cancerization in the urothelium. Receiver operating characteristic curve analysis confirmed the feasibility of cancer risk estimation to identify urothelium at the precancerous stage by DNA methylation quantification. Cancer risk estimation permutation was established using a combination of two marker CpG loci on the HOXC4, TENM3 and TLR1 genes (sensitivity and specificity 96-100%). Among them, the diagnostic impact of 10 patterns of permutation was successfully validated in the validation cohort (sensitivity and specificity 94-98%). These data suggest that cancer risk estimation using procedures such as urine tests during health checkups might become applicable for clinical use.

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Array comparative genomic hybridization analysis discloses chromosome copy number alterations as indicators of patient outcome in lymph node-negative breast cancer

Kikuchi-Koike R., Nagasaka K., Tsuda H., Ishii Y., Sakamoto M., Kikuchi Y., Fukui S., Miyagawa Y., Hiraike H., Kobayashi T., Kinoshita T., Kanai Y., Shibata T., Imoto I., Inazawa J., Matsubara O., Ayabe T.

BMC Cancer (BMC Cancer) 19 ( 1 ) 521 - 521 2019.05

Research paper (scientific journal), Joint Work

© 2019 The Author(s). Background: Patients with lymph node metastasis-negative (pN0) invasive breast cancer have favorable outcomes following initial treatment. However, false negatives which occur during routine histologic examination of lymph nodes are reported to underestimate the clinical stage of disease. To identify a high-risk group in pN0 invasive breast cancer, we examined copy number alterations (CNAs) of 800 cancer-related genes. Methods: Using array-based comparative genomic hybridization (CGH) in 51 pN0 cases (19 relapsed and 32 non-relapsed cases), the positivities of specific gene CNAs in the relapsed and non-relapsed groups were compared. An unsupervised hierarchical cluster analysis was then performed to identify case groups that were correlated with patient outcomes. Results: The cluster analysis identified three distinct clusters of cases: groups 1, 2, and 3. The major component was triple-negative cases (69%, 9 of 13) in group 1, luminal B-like (57%, 13 of 23) and HER2-overexpressing (26%, 6 of 23) subtypes in group 2, and luminal A-like subtype (60%, 9 of 15) in group 3. Among all 51 cases, those in group 1 showed significantly worse overall survival (OS) than

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Establishment of Patient-Derived Organoids and Drug Screening for Biliary Tract Carcinoma

Saito Y., Muramatsu T., Kanai Y., Ojima H., Sukeda A., Hiraoka N., Arai E., Sugiyama Y., Matsuzaki J., Uchida R., Yoshikawa N., Furukawa R., Saito H.

Cell Reports (Cell Reports) 27 ( 4 ) 1265 - 1276.e4 2019.04

Joint Work, ISSN 2211-1247

© 2019 The Author(s) Biliary tract carcinomas (BTCs) are among the most aggressive malignancies and have a poor prognosis. Here, we successfully established organoid lines derived from intrahepatic cholangiocarcinoma, gallbladder cancer, and neuroendocrine carcinoma of the ampulla of Vater. These organoids derived from BTCs were cultured stably for >1 year and closely recapitulated the histopathology, gene expression, and genetic alterations evident in the primary tumors. Gene expression profiling of the organoids revealed that SOX2 could be a potential prognostic biomarker for patients with BTC. We screened a compound library consisting of drugs used clinically for their ability to suppress organoids derived from BTCs and found that the antifungal drugs amorolfine and fenticonazole significantly suppressed the growth of organoids derived from BTCs with minimal toxicity to normal biliary epithelial cells. Patient-derived organoids may be a powerful research tool for the clarification of molecular pathogenesis and the discovery of biomarkers and therapeutic drugs for refractory cancers.

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Steatotic and nonsteatotic scirrhous hepatocellular carcinomas reveal distinct clinicopathological features

Hatano M., Ojima H., Masugi Y., Tsujikawa H., Hiraoka N., Kanai Y., Shimada K., Shinoda M., Sakamoto M.

Human Pathology (Human Pathology) 86 222 - 232 2019.04

Research paper (scientific journal), Joint Work, ISSN 00468177

© 2018 Elsevier Inc. We investigated the clinicopathological and molecular characteristics of scirrhous hepatocellular carcinoma (HCC) to elucidate its uniqueness. Samples from 120 resected HCC cases underwent immunohistochemical analysis. Tumor area containing fibrous stroma and the percentage of steatotic cells within the tumor were evaluated. In our previous report, tumors were immunohistochemically subclassified as biliary/stem cell markers–positive (B/S) (cytokeratin 19 and/or sal-like protein 4 and/or epithelial cell adhesion molecule positive), Wnt/β-catenin signaling–related markers–positive (W/B) (β-catenin and/or glutamine synthetase positive), or all markers–negative (−/−) groups. Thirty-seven cases (31%) with fibrous stroma making up ≥50% of the largest tumor area were defined as scirrhous HCC (sHCC); the other 83 cases (69%) were categorized as common HCC (cHCC). Clinicopathologically, sHCC had fewer poorly differentiated tumors (P =.037) and a higher percentage of cases with steatosis (P =.025) than cHCC. sHCC cases were further divided into two subgroups: those with ≥5% steatotic cells (steatotic sHCC) and those with <5% steatotic cells (nonsteatot

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Clinical and endoscopic findings to assist the early detection of duodenal adenoma and adenocarcinoma

Matsuzaki J., Suzuki H., Shimoda M., Mori H., Fukuhara S., Miyoshi S., Masaoka T., Iwao Y., Kanai Y., Kanai T.

United European Gastroenterology Journal (United European Gastroenterology Journal) 7 ( 2 ) 250 - 260 2019.03

Joint Work, ISSN 20506406

© Author(s) 2018. Background: Sporadic nonampullary duodenal adenocarcinoma is a rare malignant neoplasm in which poor prognosis is often associated with delayed diagnosis. Objective: A case-control study was designed to evaluate the clinical and endoscopic characteristics of patients with nonampullary duodenal epithelial tumours (NADETs). Methods: Patients with NADETs were chronologically divided into a discovery and a validation sets. Two age- and sex-matched control individuals for each case in the discovery set were randomly selected from individuals without NADET. A prediction model for the presence of NADET, constructed in the discovery set, was evaluated in the validation set. Results: In total, 368 adenomas, 81 adenocarcinomas, and 314 controls were analysed. Current smoking, Barrett oesophagus, fundic gland polyps, history of malignant disease, and absence of dyslipidaemia were independently associated with the presence of NADET. The combination of these five factors enabled significant discrimination for NADET in the bulb with a sensitivity of 0.81 in the validation set. We also showed that duodenal adenocarcinomas in the bulb had greater invasive potential than adenocar

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Prospective exosome-focused translational research for afatinib study of non-small cell lung cancer patients expressing EGFR (EXTRA study)

Okuma Y., Morikawa K., Tanaka H., Yokoyama T., Itani H., Horiuchi K., Nakagawa H., Takahashi N., Bessho A., Soejima K., Kishi K., Togashi A., Kanai Y., Ueda K., Horimoto K., Matsutani N., Seki N.

Thoracic Cancer (Thoracic Cancer) 10 ( 2 ) 395 - 400 2019.02

Joint Work, Accepted, ISSN 17597706

© 2018 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd Patients with EGFR-mutated non-small cell lung cancer (NSCLC) exhibit resistance to EGFR-tyrosine kinase inhibitors (TKIs) within 9–14 months of therapy. Recently, EGFR-mutated NSCLC has demonstrated the potential for heterogeneity; therefore, the manner of clonal heterogeneity may impact the duration of progression-free and overall survival and other parameters affecting EGFR-TKI treatment efficacy. However no predictive biomarker of these favorable treatment efficacies has been identified to date. The exosome-focused translational research for afatinib (EXTRA) study aims to identify a novel predictive biomarker and a resistance marker for afatinib by analyzing data from association studies of the clinical efficacy of afatinib and four “OMICs” (genomics, proteomics, epigenomics, and metabolomics) using peripheral blood from patients treated with afatinib. This study aims to: (i) conduct comprehensive multi-OMIC analyses in a prospective clinical trial, and (ii) focus on both sera/plasma and exosome as a source for OMIC analyses to identify a novel predictor of t

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Human amniotic fluid stem cells have a unique potential to accelerate cutaneous wound healing with reduced fibrotic scarring like a fetus

Fukutake M., Ochiai D., Masuda H., Abe Y., Sato Y., Otani T., Sakai S., Aramaki-Hattori N., Shimoda M., Matsumoto T., Miyakoshi K., Kanai Y., Kishi K., Tanaka M.

Human Cell (Human Cell) 32 ( 1 ) 51 - 63 2019.01

Joint Work, ISSN 09147470

© 2018, Japan Human Cell Society and Springer Japan KK, part of Springer Nature. Adult wound healing can result in fibrotic scarring (FS) characterized by excess expression of myofibroblasts and increased type I/type III collagen expression. In contrast, fetal wound healing results in complete regeneration without FS, and the mechanism remains unclear. Amniotic fluid cells could contribute to scar-free wound healing, but the effects of human amniotic fluid cells are not well characterized. Here, we determined the effect of human amniotic fluid stem cells (hAFS) on FS during wound healing. Human amniotic fluid was obtained by amniocentesis at 15–17 weeks of gestation. CD117-positive cells were isolated and defined as hAFS. hAFS (1 × 10 6 ) suspended in PBS or cell-free PBS were injected around wounds created in the dorsal region of BALB/c mice. Wound size was macroscopically measured, and re-epithelialization in the epidermis, granulation tissue area in the dermis and collagen contents in the regenerated wound were histologically analyzed. The ability of hAFS to engraft in the wound was assessed by tracking hAFS labeled with PKH-26. hAFS fulfilled the minimal criteria for mesen

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Primary Peritoneal Low-grade Serous Carcinoma in a Patient with Lynch Syndrome: A Case Report

Makiuchi S., Yoshida H., Ishikawa M., Kojima N., Kanai Y., Kato T.

International Journal of Gynecological Pathology (International Journal of Gynecological Pathology) 39 ( 4 ) 327 - 332 2019

Joint Work, ISSN 02771691

Copyright © 2019 The Author(s). Lynch syndrome (LS) is an autosomal dominant disease caused by a germline mutation in DNA mismatch repair genes which increases the risk of several cancers such as endometrial and colorectal cancers. However, there are only a few reports of peritoneal malignancies in patients with LS. Herein, we report the first case of a primary peritoneal lowgrade serous carcinoma in a woman with LS and provide a literature review of peritoneal malignancies in patients with LS. The patient was a 72-yr-old gravid 2 para 2 Japanese woman with a germline mutation in MLH1. She had a history of colon cancer and endometrial cancer and was treated with total hysterectomy and bilateral salpingo-oophorectomy 14 yr ago. During the follow-up, peritoneal nodules were detected by abdominal computed tomography which were surgically resected. Pathologic examination revealed a low-grade serous carcinoma with cells positive for BerEP4, MOC31, CEA, and WT-1 and negative for BAP1, PAX8, MLH1, and PMS2, by immunohistochemistry. This case report and literature review show that peritoneal low-grade serous carcinoma can occur in patients with LS and that LS-related cancers usually prece

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Prognosis method for renal cell cancer

Y Kanai, E Arai, Y Nemoto, T Yotani

US Patent App. 15/756,812 2019

Prognosis method for renal cell cancer

Y Kanai, E Arai, Y Nemoto, T Yotani

US Patent App. 15/756,812 2019

Prognosis method for renal cell cancer

Y Kanai, E Arai, Y Nemoto, T Yotani

US Patent App. 15/756,812 2019

Prognosis method for renal cell cancer

Y Kanai, E Arai, Y Nemoto, T Yotani

US Patent App. 15/756,812 2019

Physiological Functions of LMP2/β1i in the Female Reproductive System

T Hayashi, K Sano, T Ichimura, G Gur, H Aburatani, Y Kanai, D Zharhary, ...

2019

Physiological Functions of LMP2/β1i in the Female Reproductive System

T Hayashi, K Sano, T Ichimura, G Gur, H Aburatani, Y Kanai, D Zharhary, ...

2019

Assessment of homologous recombination deficiency phenotype in breast cancers in adolescents and young adults in the clinical setting

T Watanabe, T Honda, H Totsuka, M Yoshida, M Tanioka, K Shiraishi, ...

2019

Assessment of homologous recombination deficiency phenotype in breast cancers in adolescents and young adults in the clinical setting

T Watanabe, T Honda, H Totsuka, M Yoshida, M Tanioka, K Shiraishi, ...

2019

Assessment of homologous recombination deficiency phenotype in breast cancers in adolescents and young adults in the clinical setting

T Watanabe, T Honda, H Totsuka, M Yoshida, M Tanioka, K Shiraishi, ...

2019

Assessment of homologous recombination deficiency phenotype in breast cancers in adolescents and young adults in the clinical setting

T Watanabe, T Honda, H Totsuka, M Yoshida, M Tanioka, K Shiraishi, ...

2019

乳頭状腎細胞癌

Arai Eri, KANAI Yae

病理と臨床臨時増刊号「肉眼病理　症例から探る鑑別のヒント」 37 253 - 255 2019

Accepted

集合管癌

Arai Eri, KANAI Yae

病理と臨床臨時増刊号「肉眼病理　症例から探る鑑別のヒント」 37 259 - 261 2019

Accepted

精巣胚細胞腫瘍の転移

Arai Eri, KANAI Yae

病理と臨床臨時増刊号「肉眼病理　症例から探る鑑別のヒント」 37 299 - 301 2019

Accepted

精巣胚細胞腫瘍

Arai Eri, KANAI Yae

病理と臨床臨時増刊号「肉眼病理　症例から探る鑑別のヒント」 37 294 - 296 2019

Accepted

精巣悪性リンパ腫

Arai Eri, KANAI Yae

病理と臨床臨時増刊号「肉眼病理　症例から探る鑑別のヒント」 37 297 - 298 2019

Accepted

嫌色素細胞がんとオンコサイトーマ

Arai Eri, KANAI Yae

病理と臨床臨時増刊号「肉眼病理　症例から探る鑑別のヒント」 37 256 - 258 2019

Accepted

The functional analysis of miR-200 family in renal cell carcinoma

Gotoh Masahiro, Arai Eri, Yoshida Teruhiko, Kanai Yae

CANCER SCIENCE 109 446 - 446 2018.12

Joint Work, ISSN 1349-7006

Accumulation of tumor associated macrophages by miR-27b regulated CSF1 in renal cell carcinoma

Matsumoto Daichi, Tanaka Keiyu, Arai Eri, Kanai Yae, Ohta Shigeki, Kawakami Yutaka

CANCER SCIENCE 109 1192 - 1192 2018.12

Joint Work, ISSN 1349-7006

Development of gastric cancer-specific markers based on methylome analysis

Kameyama Shinichi, Arai Eri, Tian Ying, Ito Nanako, Shibuya Ayako, Taniguchi Hirokazu, Yoshida Teruhiko, Kanai Yae

CANCER SCIENCE 109 430 - 430 2018.12

Joint Work, ISSN 1349-7006

Epigenomic alterations during hepatocarcinogenesis without viral, alcoholic and fatty liver injury

Makiuchi Satomi, Arai Eri, Kuramoto Junko, Tian Ying, Fukamachi Yukihiro, Takahashi Yoriko, Hiraoka Nobuyoshi, Yoshida Teruhiko, Kanai Yae

CANCER SCIENCE 109 1304 - 1304 2018.12

Joint Work, ISSN 1349-7006

Genome-wide DNA methylation profile of young-onset endometrial cancer

Makabe Takeshi, Arai Eri, Hirasawa Akira, Yamagami Wataru, Susumu Nobuyuki, Aoki Daisuke, Kanai Yae

CANCER SCIENCE 109 1304 - 1304 2018.12

Joint Work, ISSN 1349-7006

Feasibility of methylome analysis using small amounts of genomic DNA from formalin-fixed paraffin-embedded tissue.

Ohara K, Arai E, Takahashi Y, Fukamachi Y, Ito N, Maeshima AM, Fujimoto H, Yoshida T, Kanai Y.

Pathol Int 68 ( 11 ) 633 - 635 2018.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 1320-5463

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GENOME-WIDE DNA METHYLATION ANALYSIS IN YOUNG-ONSET ENDOMETRIAL CANCER

Makabe T., Arai E., Hirasawa A., Yamagami W., Nobuyuki S., Aoki D., Kanai Y.

INTERNATIONAL JOURNAL OF GYNECOLOGICAL CANCER 28 79 - 79 2018.09

Joint Work, ISSN 1048-891X

Deleterious Pulmonary Surfactant System Gene Mutations in Lung Adenocarcinomas Associated With Usual Interstitial Pneumonia

Honda Takayuki, Sakashita Hiroyuki, Masai Kyohei, Totsuka Hirohiko, Motoi Noriko, Kobayashi Masashi, Akashi Takumi, Mimaki Sachiyo, Tsuchihara Katsuya, Chiku Suenori, Shiraishi Kouya, Shimada Yoko, Otsuka Ayaka, Kanai Yae, Okubo Kenichi, Watanabe Shun-ichi, Tsuta Koji, Inase Naohiko, Kohno Takashi

JCO PRECISION ONCOLOGY (JCO Precision Oncology) 2 1 - 24 2018.08

Joint Work

© 2019 American Society of Clinical Oncology. Purpose Usual interstitial pneumonia (UIP) is a risk factor for lung carcinogenesis. This study was performed to characterize mutagenesis and mutational target genes underlying lung carcinogenesis in patients with UIP. Patients and Methods A cohort of 691 Japanese patients with lung adenocarcinoma (LADC), of whom 54 had UIP and 637 did not, was studied for driver oncogene aberrations. Whole-exome analysis was performed for 296 cases, including 51 with UIP, to deduce mutagenic processes and identify commonly affected genes. Logistic regression analysis was used to detect associations of gene aberrations with clinicopathological factors. Results The EGFR mutation was markedly less prevalent in patients with LADC with UIP than in those without (1.9% [one of 54] v. 49.9% [318 of 637]; P <.001), even in heavy smokers (25.3% [38 of 150] of patients with > 40 pack-years; P <.001). Mutational signature analysis indicated that UIP-positive LADCs develop through accumulation of single-nucleotide and indel mutations caused by smoking. Pulmonary surfactant system genes (PSSGs) NKX2-1/TTF1, SFTPA1, SFTPA2, SFTPB, and SFTPC were identified as targets for mutations (preferentially indels), and mutations were specifically associated with shorter overall survival of patients with UIP-positive LADC, independent of pathologic stage (hazard ratio, 4.9; 95% CI, 1.7 to 14.4; P =.0037). Conclusion LADCs with UIP develop through mutational events caused by smoking, independently of EGFR mutation. PSSGs were identified as a mutational target and as a novel prognostic factor in UIP-positive LADC. PSSG deficiency might increase the malignancy of tumor cells by increasing the tumor-promoting effects of UIP.

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Intermittent Exposure to Cigarette Smoke Increases Lung Tumors and the Severity of Emphysema More than Continuous Exposure

Kameyama, Naofumi, Chubachi, Shotaro, Hegab, Ahmed E., Yasuda, Hiroyuki, Kagawa, Shizuko, Tsutsumi, Akihiro, Fukunaga, Koichi, Shimoda, Masayuki, Kanai, Yae, Soejima, Kenzo, Betsuyaku, Tomoko

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY (American Journal of Respiratory Cell and Molecular Biology) 59 ( 2 ) 179 - 188 2018.08

Research paper (scientific journal), Single Work, ISSN 1044-1549

Copyright © 2018 by the American Thoracic Society. Lung cancer and chronic obstructive pulmonary disease are leading causes of morbidity and mortality worldwide, and cigarette smoking is a main risk factor for both. The presence of emphysema, an irreversible lung disease, further raises the risk of lung cancer in patients with chronic obstructive pulmonary disease. The mechanisms involved in smoke-induced tumorigenesis and emphysema are not fully understood, attributable to a lack of appropriate animal models. Here, we optimized a model of cigarette smoke (CS)–induced lung cancer and emphysema in A/J mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a potent carcinogen. We investigated whether variations in CS exposure patterns with the same total amount and duration of exposure affect tumorigenesis and/or development of emphysema. Continuous CS exposure for 3 months significantly suppressed 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone–induced development of adenomas and adenocarcinomas; however, emphysema independently developed during this period. Surprisingly, intermittent CS exposure increased the severity of emphysema and resulted in a higher incidence o

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Establishment and long-term in vitro culture of organoids derived from human biliary tract carcinomas

Saito Yoshimasa, Muramatsu Toshihide, Sugiyama Yuko, Uchida Ryoei, Furukawa Ryo, Yoshikawa Nao, Yamaguchi Tomoko, Kanai Yae, Saito Hidetsugu

CANCER RESEARCH 78 ( 13 ) 2018.07

Joint Work, ISSN 0008-5472

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A paradoxical thrombogenic mutation in factor II at the target site of arthropod bleeding toxin.

Takenouchi T, Shimada H, Uehara T, Kanai Y, Takahashi T, Kosaki K.

Eur J Med Genet (European Journal of Medical Genetics) 62 ( 2 ) 93 - 95 2018.06

Research paper (scientific journal), Single Work, Accepted, ISSN 17697212

© 2018 Loss-of-function mutations in coagulation cascade proteins lead to bleeding diasthesis. In contrast, gain-of-function mutations in these proteins, which are exceptionally rare, lead to hereditary thrombosis. This is best exemplified by Factor V (i.e., Factor V Leiden) and Factor II (i.e., p.Arg596Leu). Here, we report a family with hereditary thrombosis. The proposita presented with cerebral venous thrombosis accompanied by infarction at the age of 12 years. Despite anticoagulation therapy with oral warfarin, she later developed deep venous thrombosis in her hepatic portal veins at the age of 27 years. A medical exome analysis identified a de novo heterozygous mutation p.Tyr434His in Factor II, which was segregated within the family. A retrospective molecular diagnosis was made using a preserved surgical specimen from the proposita's mother, who had died 10 years earlier. The p.Tyr434 residue, which was substituted in the presently reported family, was located in “exosite I” of thrombin, a critical recognition site for fibrinogen, protein C, and thrombin aptamer. Therefore, the mutant thrombin likely exerted its thrombophilic effect by altering the affinity of thrombin

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Semiquantitative Evaluation of Muscle Repair by Diffusion Tensor Imaging in Mice.

Hata J, Mizuno S, Haga Y, Shimoda M, Kanai Y, Chiba K, Okano H, Nakamura M, Horiuchi K.

JBMR Plus (JBMR Plus) 2 ( 4 ) 227 - 234 2018.05

Research paper (scientific journal), Joint Work

Muscle injury is one of the most common traumas in orthopedic and sports medicine. However, there are only a few treatment options with marginal clinical benefits for this condition. Muscle repair after injury involves multiple and complex processes, such as the inflammation phase, regeneration phase, and remodeling phase. To develop a treatment modality and to examine the efficacy of novel interventions and agents for patients with muscle injuries, it is essential to establish a reliable and sensitive method to monitor the changes in muscle structure and status during muscle repair. Diffusion-weighted magnetic resonance imaging has been widely used to assess the diffusivity of water molecules in tissue. When it is used in combination with diffusion tensor imaging (DTI), the microstructure of muscle tissue can be indirectly depicted. In the present study, we evaluated the time-course changes in the diffusivity and anisotropy in muscles by DTI and histology after injury in mice. We found that the diffusivity and anisotropy exhibit distinct kinetics during muscle repair and that these kinetics were significantly altered in mutant mice with a defect in muscle regeneration. Our data show that muscle repair processes can be readily evaluated and monitored by DTI technique and suggest that DTI can be clinically applied for assessing muscle injury and repair in humans. © 2018 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

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

Yotani, Takuya, Yamada, Yuriko, Arai, Eri, Tian, Ying, Gotoh, Masahiro, Komiyama, Motokiyo, Fujimoto, Hiroyuki, Sakamoto, Michiie, Kanai, Yae

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

Research paper (scientific journal), Single Work, ISSN 1349-7006

© 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 methylat

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DBTSS/DBKERO for integrated analysis of transcriptional regulation

Suzuki, Ayako, Kawano, Shin, Mitsuyama, Toutai, Suyama, Mikita, Kanai, Yae, Shirahige, Katsuhiko, Sasaki, Hiroyuki, Tokunaga, Katsushi, Tsuchihara, Katsuya, Sugano, Sumio, Nakai, Kenta, Suzuki, Yutaka

NUCLEIC ACIDS RESEARCH (Nucleic Acids Research) 46 ( D1 ) D229 - D238 2018.01

Research paper (scientific journal), Single Work, ISSN 0305-1048

© 2017 The Author(s). DBTSS (Database of Transcriptional Start Sites)/DBKERO (Database of Kashiwa Encyclopedia for human genome mutations in Regulatory regions and their Omics contexts) is the database originally initiated with the information of transcriptional start sites and their upstream transcriptional regulatory regions. In recent years, we updated the database to assist users to elucidate biological relevance of the human genome variations or somatic mutations in cancers which may affect the transcriptional regulation. In this update, we facilitate interpretations of disease associated genomic variation, using the Japanese population as a model case. We enriched the genomic variation dataset consisting of the 13,368 individuals collected for various genome-wide association studies and the reference epigenome information in the surrounding regions using a total of 455 epigenome datasets (four tissue types from 67 healthy individuals) collected for the International Human Epigenome Consortium (IHEC). The data directly obtained from the clinical samples was associated with that obtained from various model systems, such as the drug perturbation datasets using cultured cancer c

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Integrated analysis of genetic and epigenetic alterations in gastric carcinomas

Yang Menghan, Arai Eri, Sakamoto Hiromi, Totsuka Hirohiko, Taniguchi Hirokazu, Katai Hitoshi, Yoshida Teruhiko, Kanai Yae

CANCER SCIENCE 109 584 - 584 2018.01

ISSN 1349-7006

Genome-wide DNA methylation analysis in non-alcoholic steatohepatitis-related hepatocellular carcinomas

Tian Ying, Arai Eri, Kuramoto Kunko, Sukeda Aoi, Ojima Hidenori, Yoshida Teruhiko, Kanai Yae

CANCER SCIENCE 109 238 - 238 2018.01

Joint Work, ISSN 1349-7006

Carcinogenic risk estimation based on genome-wide DNA methylation analysis during urothelial carcinogenesis

Tsumura Koji, Arai Eri, Tian Ying, Fujimoto Hiroyuki, Nakagawa Tohru, Homma Yukio, Yoshida Teruhiko, Kanai Yae

CANCER SCIENCE 109 49 - 49 2018.01

Joint Work, ISSN 1349-7006

Formalin-fixed and paraffin-embedded tissue samples are available for genome-wide DNA methylation analysis

Ohara Kentaro, Arai Eri, Fukamachi Yukihiro, Takahashi Yoriko, Komiyama Motokiyo, Fujimoto Hiroyuki, Yoshida Teruhiko, Kanai Yae

CANCER SCIENCE 109 239 - 239 2018.01

Joint Work, ISSN 1349-7006

Genome-wide DNA methylation analysis during lung adenocarcinogenesis

Hamada Kenichi, Arai Eri, Kohno Takashi, Takahashi Yoriko, Fukamachi Yukihiro, Tsuta Koji, Watanabe Shunichi, Yoshida Teruhiko, Asamura Hisao, Kanai Yae

CANCER SCIENCE 109 239 - 239 2018.01

Joint Work, ISSN 1349-7006

The Japanese Society of Pathology Guidelines on the handling of pathological tissue samples for genomic research: Standard operating procedures based on empirical analyses.

Kanai Y, Nishihara H, Miyagi Y, Tsuruyama T, Taguchi K, Katoh H, Takeuchi T, Gotoh M, Kuramoto Y, Arai E, Ojima H, Shibuya A, Yoshida T, Akahane T, Kasajima R, Morita K, Inazawa J, Sasaki T, Fukayama M, Oda Y.

Pathol Int (Pathology International) 68 ( 2 ) 63 - 90 2018

Research paper (scientific journal), Joint Work, ISSN 13205463

© 2018 The Authors. Pathology International published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd Genome research using appropriately collected pathological tissue samples is expected to yield breakthroughs in the development of biomarkers and identification of therapeutic targets for diseases such as cancers. In this connection, the Japanese Society of Pathology (JSP) has developed “The JSP Guidelines on the Handling of Pathological Tissue Samples for Genomic Research” based on an abundance of data from empirical analyses of tissue samples collected and stored under various conditions. Tissue samples should be collected from appropriate sites within surgically resected specimens, without disturbing the features on which pathological diagnosis is based, while avoiding bleeding or necrotic foci. They should be collected as soon as possible after resection: at the latest within about 3 h of storage at 4°C. Preferably, snap-frozen samples should be stored in liquid nitrogen (about −180°C) until use. When intending to use genomic DNA extracted from formalin-fixed paraffin-embedded tissue, 10% neutral buffered formalin should be used. Insufficient fi

Epigenome mapping of human normal purified hepatocytes: personal epigenome variation and genome-epigenome correlation.

Arai E, Miura F, Totoki Y, Yamashita S, Tian Y, Gotoh M, Ojima H, Nakagawa H, Takahashi Y, Nakamura H, Hama N, Kato M, Kimura H, Suzuki Y, Ito T, Shibata T, Kanai Y.

Epigenomics (Epigenomics) 10 ( 7 ) 955 - 979 2018

Research paper (scientific journal), Single Work, ISSN 17501911

© 2018 Yae Kanai. Aim: The aim of this study was to reveal the epigenome landscape of human normal hepatocytes. Materials & methods: Cells purified from partial hepatectomy specimens of Japanese patients were subjected to whole-genome bisulfite sequencing using postbisulfite adaptor tagging, chromatin immunoprecipitation sequencing, RNA sequencing and whole-genome sequencing. Results: CHG and CHH methylations were inversely associated with gene expression. Histone modification profiles of personal differentially methylated regions (pDMRs) differed considerably among samples. pDMRs were observed around the transcription start sites of genes whose expression is reportedly regulated by CpG methylation. pDMRs were frequently observed in the vicinity of single-nucleotide variations and insertions/deletions. Conclusion: Genetic variations may induce epigenetic variations, generating individual differences in the phenotypes of normal hepatocytes through variations in expression.

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Induction of differentiation of intrahepatic cholangiocarcinoma cells to functional hepatocytes using an organoid culture system.

Saito Y, Nakaoka T, Muramatsu T, Ojima H, Sukeda A, Sugiyama Y, Uchida R, Furukawa R, Kitahara A, Sato T, Kanai Y, Saito H.

Sci Rep (Scientific Reports) 8 ( 1 ) 2821 2018

Research paper (scientific journal), Single Work, Accepted, ISSN 2045-2322

© 2018 The Author(s). Intrahepatic cholangiocarcinoma (IHCC) is a highly aggressive malignancy with a poor prognosis. It is thought to originate from cholangiocytes, which are the component cells of intrahepatic bile ducts. However, as patients with viral hepatitis often develop IHCC, it has been suggested that transformed hepatocytes may play a role in IHCC development. To investigate whether IHCC cells can be converted to functional hepatocytes, we established organoids derived from human IHCC and cultured them under conditions suitable for hepatocyte differentiation. IHCC organoids after hepatocyte differentiation acquired functions of mature hepatocytes such as albumin secretion, bile acid production and increased CYP3A4 activity. Studies using a mouse model of IHCC indicate that Wnt3a derived from macrophages recruited upon inflammation in the liver may promote the malignant transformation of hepatocytes to IHCC cells. The results of the present study support the recently proposed hypothesis that IHCC cells are derived from hepatocytes.

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ADAM9 is over-expressed in human ovarian clear cell carcinomas and suppresses cisplatin-induced cell death.

Ueno M, Shiomi T, Mochizuki S, Chijiiwa M, Shimoda M, Kanai Y, Kataoka F, Hirasawa A, Susumu N, Aoki D, Okada Y.

Cancer Sci (Cancer Science) 109 ( 2 ) 471 - 482 2018

Research paper (scientific journal), Single Work, Accepted, ISSN 13479032

© 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. ADAMs (a disintegrin and metalloproteinases) are involved in various biological events such as cell adhesion, migration and invasion, membrane protein shedding and proteolysis. However, there have been no systematic studies on the expression of ADAMs in human ovarian carcinomas. We therefore examined mRNA expression of all the proteolytic ADAM species including ADAM8, 9, 10, 12, 15, 17, 19, 20, 21, 28, 30, 33 and ADAMDEC1 in human ovarian carcinomas, and found that prototype membrane-anchored ADAM9m, but not secreted isoform ADAM9s, is significantly over-expressed in carcinomas than in control non-neoplastic ovarian tissue. Among the histological subtypes of serous, endometrioid, mucinous and clear cell carcinomas, ADAM9m expression was highest in clear cell carcinomas. Immunohistochemistry showed that all the clear cell carcinoma samples displayed ADAM9m primarily on the carcinoma cell membrane. By immunoblotting, ADAM9m was detected mainly in an active form in the clear cell carcinoma tissues. When two clear cell carcinoma cell lines (RMG-I and TOV21G cell

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511 IDENTIFICATION OF CYP2A6 GENE RELATED TO INVASIVE BLADDER CARCINOGENESIS

K Kanemoto, K Fukuta, K Yamada, N Kawai, K Tozawa, Y Kanai, ...

JOURNAL OF UROLOGY 200 (3), e208-e208 2018

Somatic mutations in IFN-γ-Signal molecules in human uterine leiomyosarcoma

T Hayashi, T Ichimura, M Kasai, K Sano, S Tonegawa, Y Kanai, ...

2018

Somatic mutations in IFN-γ-Signal molecules in human uterine leiomyosarcoma

T Hayashi, T Ichimura, M Kasai, K Sano, S Tonegawa, Y Kanai, ...

2018

Somatic mutations in IFN-γ-Signal molecules in human uterine leiomyosarcoma

T Hayashi, T Ichimura, M Kasai, K Sano, S Tonegawa, Y Kanai, ...

2018

Risk estimation for hepatocellular carcinoma by DNA methylation analysis using high performance liquid chromatography

E Sunamura, T Yotani, F Arai, Y Tian, A Skeda, H Ojima, T Yoshida, ...

CANCER SCIENCE 109, 243-244 2018

Risk estimation for hepatocellular carcinoma by DNA methylation analysis using high performance liquid chromatography

E Sunamura, T Yotani, F Arai, Y Tian, A Skeda, H Ojima, T Yoshida, ...

CANCER SCIENCE 109, 243-244 2018

Risk estimation for hepatocellular carcinoma by DNA methylation analysis using high performance liquid chromatography

E Sunamura, T Yotani, F Arai, Y Tian, A Skeda, H Ojima, T Yoshida, ...

CANCER SCIENCE 109, 243-244 2018

Risk estimation for hepatocellular carcinoma by DNA methylation analysis using high performance liquid chromatography

E Sunamura, T Yotani, F Arai, Y Tian, A Skeda, H Ojima, T Yoshida, ...

CANCER SCIENCE 109, 243-244 2018

Patient Background and Responses in Providing Informed Consent for the National Cancer Center Biobank Project.

T Yamane, S Yamagami, N Yamashita, T Yoshida, K Tobinai, Y Kanai, ...

CANCER SCIENCE 109, 789-789 2018

Patient Background and Responses in Providing Informed Consent for the National Cancer Center Biobank Project.

T Yamane, S Yamagami, N Yamashita, T Yoshida, K Tobinai, Y Kanai, ...

CANCER SCIENCE 109, 789-789 2018

Patient Background and Responses in Providing Informed Consent for the National Cancer Center Biobank Project.

T Yamane, S Yamagami, N Yamashita, T Yoshida, K Tobinai, Y Kanai, ...

CANCER SCIENCE 109, 789-789 2018

Patient Background and Responses in Providing Informed Consent for the National Cancer Center Biobank Project.

T Yamane, S Yamagami, N Yamashita, T Yoshida, K Tobinai, Y Kanai, ...

CANCER SCIENCE 109, 789-789 2018

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

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

Cancer science 109 (5), 1690-1700 2018

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

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

Cancer science 109 (5), 1690-1700 2018

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

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

Cancer science 109 (5), 1690-1700 2018

Method for determining prognosis of renal cell carcinoma

Y Kanai, E Arai, Y Nemoto, T Yotani

US Patent App. 16/000,532 2018

Method for determining prognosis of renal cell carcinoma

Y Kanai, E Arai, Y Nemoto, T Yotani

US Patent App. 16/000,532 2018

Method for determining prognosis of renal cell carcinoma

Y Kanai, E Arai, Y Nemoto, T Yotani

US Patent App. 16/000,532 2018

Method for determining prognosis of renal cell carcinoma

Y Kanai, E Arai, Y Nemoto, T Yotani

US Patent App. 16/000,532 2018

Feasibility of methylome analysis using small amounts of genomic DNA from formalin‐fixed paraffin‐embedded tissue

K Ohara, E Arai, Y Takahashi, Y Fukamachi, N Ito, AM Maeshima, ...

Pathology international 68 (11), 633-635 2018

Feasibility of methylome analysis using small amounts of genomic DNA from formalin‐fixed paraffin‐embedded tissue

K Ohara, E Arai, Y Takahashi, Y Fukamachi, N Ito, AM Maeshima, ...

Pathology international 68 (11), 633-635 2018

Feasibility of methylome analysis using small amounts of genomic DNA from formalin‐fixed paraffin‐embedded tissue

K Ohara, E Arai, Y Takahashi, Y Fukamachi, N Ito, AM Maeshima, ...

Pathology international 68 (11), 633-635 2018

Epigenome mapping of human normal purified hepatocytes: personal epigenome variation and genome–epigenome correlation

E Arai, F Miura, Y Totoki, S Yamashita, Y Tian, M Gotoh, H Ojima, ...

Epigenomics 10 (7), 955-979 2018

Epigenome mapping of human normal purified hepatocytes: personal epigenome variation and genome–epigenome correlation

E Arai, F Miura, Y Totoki, S Yamashita, Y Tian, M Gotoh, H Ojima, ...

Epigenomics 10 (7), 955-979 2018

Epigenome mapping of human normal purified hepatocytes: personal epigenome variation and genome–epigenome correlation

E Arai, F Miura, Y Totoki, S Yamashita, Y Tian, M Gotoh, H Ojima, ...

Epigenomics 10 (7), 955-979 2018

ADAM9 is over‐expressed in human ovarian clear cell carcinomas and suppresses cisplatin‐induced cell death

M Ueno, T Shiomi, S Mochizuki, M Chijiiwa, M Shimoda, Y Kanai, ...

Cancer science 109 (2), 471-482 2018

ADAM9 is over‐expressed in human ovarian clear cell carcinomas and suppresses cisplatin‐induced cell death

M Ueno, T Shiomi, S Mochizuki, M Chijiiwa, M Shimoda, Y Kanai, ...

Cancer science 109 (2), 471-482 2018

ADAM9 is over‐expressed in human ovarian clear cell carcinomas and suppresses cisplatin‐induced cell death

M Ueno, T Shiomi, S Mochizuki, M Chijiiwa, M Shimoda, Y Kanai, ...

Cancer science 109 (2), 471-482 2018

511 IDENTIFICATION OF CYP2A6 GENE RELATED TO INVASIVE BLADDER CARCINOGENESIS

K Kanemoto, K Fukuta, K Yamada, N Kawai, K Tozawa, Y Kanai, ...

JOURNAL OF UROLOGY 200 (3), e208-e208 2018

511 IDENTIFICATION OF CYP2A6 GENE RELATED TO INVASIVE BLADDER CARCINOGENESIS

K Kanemoto, K Fukuta, K Yamada, N Kawai, K Tozawa, Y Kanai, ...

JOURNAL OF UROLOGY 200 (3), e208-e208 2018

511 IDENTIFICATION OF CYP2A6 GENE RELATED TO INVASIVE BLADDER CARCINOGENESIS

K Kanemoto, K Fukuta, K Yamada, N Kawai, K Tozawa, Y Kanai, ...

JOURNAL OF UROLOGY 200 (3), e208-e208 2018

Somatic mutations in IFN-γ-Signal molecules in human uterine leiomyosarcoma

T Hayashi, T Ichimura, M Kasai, K Sano, S Tonegawa, Y Kanai, ...

2018

SPECIFIC PREDICTION OF DUODENAL TUMOR USING DEMOGRAPHIC AND ENDOSCOPIC CHARACTERISTICS: AN AGE-AND SEX-MATCHED CASE-CONTROL STUDY

H Suzuki, J Matsuzaki, M Shimoda, H Mori, S Fukuhara, Y Kanai, T Kanai

Gastroenterology 154 (6), S664-S665 2018

SPECIFIC PREDICTION OF DUODENAL TUMOR USING DEMOGRAPHIC AND ENDOSCOPIC CHARACTERISTICS: AN AGE-AND SEX-MATCHED CASE-CONTROL STUDY

H Suzuki, J Matsuzaki, M Shimoda, H Mori, S Fukuhara, Y Kanai, T Kanai

Gastroenterology 154 (6), S664-S665 2018

SPECIFIC PREDICTION OF DUODENAL TUMOR USING DEMOGRAPHIC AND ENDOSCOPIC CHARACTERISTICS: AN AGE-AND SEX-MATCHED CASE-CONTROL STUDY

H Suzuki, J Matsuzaki, M Shimoda, H Mori, S Fukuhara, Y Kanai, T Kanai

Gastroenterology 154 (6), S664-S665 2018

SPECIFIC PREDICTION OF DUODENAL TUMOR USING DEMOGRAPHIC AND ENDOSCOPIC CHARACTERISTICS: AN AGE-AND SEX-MATCHED CASE-CONTROL STUDY

H Suzuki, J Matsuzaki, M Shimoda, H Mori, S Fukuhara, Y Kanai, T Kanai

Gastroenterology 154 (6), S664-S665 2018

Hyaluronan binding protein involved in hyaluronan depolymerization(HYBID)はヒアルロン酸代謝を介して内軟骨性骨形成を制御する

下田将之, 吉田浩之, 水野早希子, 弘實透, 堀内圭輔, 吉野雄太, 原英彰, 金井弥栄, 井上紳太郎, 石島旨章, 岡田保典

日本結合組織学会学術大会プログラム・抄録集 (日本結合組織学会) 49回 42 - 42 2017.06

Accepted

長管骨形成に関わるHYBIDの役割解析

下田将之, 吉田浩之, 水野早希子, 堀内圭輔, 吉野雄太, 原英彰, 金井弥栄, 井上紳太郎, 石島旨章, 岡田保典

日本病理学会会誌 ((一社)日本病理学会) 106 ( 1 ) 307 - 307 2017.03

Accepted, ISSN 0300-9181

Genes involved in development and differentiation are commonly methylated in cancers derived from multiple organs: A single-institutional methylome analysis using 1007 tissue specimens.

Ohara K, Arai E, Takahashi Y, Ito N, Shibuya A, Tsuta K, Kushima R, Tsuda H, Ojima H, Fujimoto H, Watanabe SI, Katai H, Kinoshita T, Shibata T, Kohno T, Kanai Y.

Carcinogenesis (OXFORD UNIV PRESS) 38 ( 3 ) 241 - 251 2017

Accepted, ISSN 01433334

The aim of this study was to clarify the significance of DNA methylation alterations shared by cancers derived from multiple organs. We analyzed single-institutional methylome data by single-CpG-resolution Infinium assay for 1007 samples of non-cancerous tissue (N) and corresponding cancerous tissue (T) obtained from lung, stomach, kidney, breast and liver. Principal component analysis revealed that N samples of each organ showed distinct DNA methylation profiles, DNA methylation profiles of N samples of each organ being inherited by the corresponding T samples and DNA methylation profiles of T samples being more similar to those of N samples in the same organ than those of T samples in other organs. In contrast to such organ and/or carcinogenetic factor-specificity of DNA methylation profiles, when compared with the corresponding N samples, 231 genes commonly showed DNA hypermethylation in T samples in four or more organs. Gene ontology enrichment analysis showed that such commonly methylated genes were enriched among '' transcriptional factors '' participating in development and/or differentiation, which reportedly show bivalent histone modification in embryonic stem cells. Pyrosequencing and quantitative reverse transcription-PCR revealed an inverse correlation between DNA methylation levels and mRNA expression levels of representative commonly methylated genes, such as ALX1, ATP8A2, CR1 and EFCAB1, in tissue samples. These data suggest that disruption of the differentiated state of precancerous cells via alterations of expression, independent of differences in organs and/or carcinogenetic factors, may be a common feature of DNA methylation alterations during carcinogenesis in multiple organs.

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Liver atrophy after percutaneous transhepatic portal embolization occurs in two histological phases: Hepatocellular atrophy followed by apoptosis.

Iwao Y, Ojima H, Kobayashi T, Kishi Y, Nara S, Esaki M, Shimada K, Hiraoka N, Tanabe M, Kanai Y.

World J Hepatol (Baishideng Publishing Group Co) 9 ( 32 ) 1227 - 1238 2017

Accepted, ISSN 1948-5182

AIM To clarify the histological changes associated with liver atrophy after percutaneous transhepatic portal embolization (PTPE) in pigs and humans. METHODS As a preliminary study, we performed pathological examinations of liver specimens from five pigs that had undergone PTPE in a time-dependent model of liver atrophy. In specimens from embolized lobes (EMB) and nonembolized lobes (controls), we measured the portal vein to central vein distance (PV-CV), the area and number of hepatocytes per lobule, and apoptotic activity using the terminal deoxynucleotidyl transferase dUTP nickend labeling assay. Immunohistochemical reactivities were evaluated for light chain 3 (LC3) and lysosomal-associated membrane protein 2 (LAMP2) as autophagy markers and for glutamine synthetase and cytochrome P450 2E1 (CYP2E1) as metabolic zonation markers. Samples from ten human livers taken 20-36 d after PTPE were similarly examined. RESULTS PV-CVs and lobule areas did not differ between EMB and controls at day 0, but were lower in EMB than in controls at weeks 2, 4, and 6 (P ≤ 0.001). Hepatocyte numbers were not significantly reduced in EMB at day 0 and week 2 but were reduced at weeks 4 and 6 (P ≤ 0.05). Apoptotic activity was higher in EMB than in controls at day 0 and week 4. LC3 and LAMP2 staining peaked in EMB at week 2, with no significant difference between EMB and controls at weeks 4 and 6. Glutamine synthetase and CYP2E1 zonation in EMB at weeks 2, 4, and 6 were narrower than those in controls. Human results were consistent with those of porcine specimens. CONCLUSION The mechanism of liver atrophy after PTPE has two histological phases: Hepatocellular atrophy is likely caused by autophagy in the first 2 wk and apoptosis thereafter.

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Inhibition of PDGFR signaling prevents muscular fatty infiltration after rotator cuff tear in mice.

Shirasawa H, Matsumura N, Shimoda M, Oki S, Yoda M, Tohmonda T, Kanai Y, Matsumoto M, Nakamura M, Horiuchi K.

Sci Rep (NATURE PUBLISHING GROUP) 7 41552 2017

Accepted, ISSN 2045-2322

Fatty infiltration in muscle is often observed in patients with sizable rotator cuff tear (RCT) and is thought to be an irreversible event that significantly compromises muscle plasticity and contraction strength. These changes in the mechanical properties of the affected muscle render surgical repair of RCT highly formidable. Therefore, it is important to learn more about the pathology of fatty infiltration to prevent this undesired condition. In the present study, we aimed to generate a mouse model that can reliably recapitulate some of the important characteristics of muscular fatty infiltration after RCT in humans. We found that fatty infiltration can be efficiently induced by a combination of the following procedures: denervation of the suprascapular nerve, transection of the rotator cuff tendon, and resection of the humeral head. Using this model, we found that platelet-derived growth factor receptor-alpha (PDGFR alpha)-positive mesenchymal stem cells are induced after this intervention and that inhibition of PDGFR signaling by imatinib treatment can significantly suppress fatty infiltration. Taken together, the present study presents a reliable fatty infiltration mouse model and suggests a key role for PDGFR alpha-positive mesenchymal stem cells in the process of fatty infiltration after RCT in humans.

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The genomic and epigenomic landscape in thymic carcinoma.

Saito M, Fujiwara Y, Asao T, Honda T, Shimada Y, Kanai Y, Tsuta K, Kono K, Watanabe S, Ohe Y, Kohno T. >

Carcinogenesis (OXFORD UNIV PRESS) 38 ( 11 ) 1084 - 1091 2017

Accepted, ISSN 01433334

Thymic carcinoma (TC) is a rare cancer whose genomic features have been examined in only a limited number of patients of European descent. Here, we characterized both genomic and epigenomic aberrations by whole exome sequencing, RNA sequencing, methylation array and copy number analyses in TCs from Asian patients and compared them with those in TCs from USA/European patients. Samples analyzed were 10 pairs of snap-frozen surgical specimens of cancerous and non-cancerous thymic tissue. All 10 cases were Japanese patients treated at the National Cancer Center Hospital, Japan, between 1994 and 2010. Mutational signature analysis indicated that the accumulation of age-related mutations drive TC development. We identified recurrent somatic mutations in TET2, CYLD, SETD2, TP53, FBXW7, HRAS and RB1, and no mutations in GTF2I, supporting the hypothesis that TC and thymoma are distinguishable by their genetic profiles. TCs with TET2 mutations had more hypermethylated genes than those without, and hyper-methylation was associated with downregulation of gene expression. Focal genome copy number gains, associated with elevated gene expression, were observed at the KIT (which is known to drive thymic carcinogenesis) and AHNAK2 gene loci. Taken together, the results suggest that the molecular processes leading to TC depend on the accumulation of genetic and epigenetic aberrations. In addition, epigenetic dysregulation as a result of the TET2 mutation was observed in a subset of TCs.

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Hyaluronan-binding protein involved in hyaluronan depolymerization controls endochondral ossification through hyaluronan metabolism.

Shimoda M, Yoshida H, Mizuno S, Hirozane T, Horiuchi K, Yoshino Y, Hara H, Kanai Y, Inoue S, Ishijima M, Okada Y.

Am J Pathol (ELSEVIER SCIENCE INC) 187 ( 5 ) 1162 - 1176 2017

Accepted, ISSN 00029440

Hyaluronan (HA) plays an important role in the development and maintenance of tissues, and its degradation is implicated in many pathologic conditions. We recently reported that HA-binding protein involved in HA depolymerization (CEMIP; alias HYBID/KIAA1199) is a key molecule in HA depolymerization, but its developmental and pathologic functions remain elusive. We generated Hybid-deficient mice using the Cre/Locus of crossover in P1 (LoxP) system and analyzed their phenotypes. Hybiddeficient mice were viable and fertile, but their adult long bones were shorter than those of wild-type animals. Hybid-deficient mice showed lengthening of hypertrophic zone in the growth plate until 4 weeks after birth. There were fewer capillaries and osteoclasts at the chondroosseous junction in the Hybid-deficient mice compared with the wild-type mice. In situ hybridization demonstrated that Hybid was expressed by hypertrophic chondrocytes at the chondroosseous junction. Cultured primary chondrocytes expressed higher levels of Hybid than did osteoblasts or osteoclasts, and the Hybid expression in the chondrocytes was up-regulated after maturation to hypertrophic chondrocytes.high-molecular-weight HA was accumulated in the lengthened hypertrophic zone in Hybid-deficient mice. In addition, high-molecular-weight HA significantly reduced cell growth and tube formation in vascular endothelial growth factor-stimulated or -nonstimulated endothelial cells. HA metabolism by HYBID is involved in endochondraL ossification during postnatal development by modulation of angiogenesis and osteoclast recruitment at the chondroosseous junction.

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Cluster microRNAs miR-194 and miR-215 suppress the tumorigenicity of intestinal tumor organoids.

Nakaoka T, Saito Y, Shimamoto Y, Muramatsu T, Kimura M, Kanai Y, Saito H.

Cancer Sci (Blackwell Publishing Ltd) 108 ( 4 ) 678 - 684 2017

Accepted, ISSN 13479032

Tumor stem cells with self-renewal and multipotent capacity play critical roles in the initiation and progression of cancer. Recently, a new 3-D culture system known as organoid culture has been developed, allowing Lgr5-positive stem cells to form organoids that resemble the properties of original tissues. Here we established organoids derived from intestinal tumors of Apcmin/+ mice and normal intestinal epithelia of C57BL/6J mice and investigated the roles of microRNA (miRNA) in intestinal tumor organoids. The results of microarray analyses revealed that expression of the cluster miRNAs, miR-194 and miR-215 was markedly suppressed in intestinal tumor organoids in comparison with organoids derived from normal intestinal epithelia. Enforced expression of miR-194 resulted in inhibition of E2f3, a positive regulator of the cell cycle and growth suppression of intestinal tumor organoids. In addition, enforced expression of miR-215 suppressed the cancer stem cell signature through downregulation of intestinal stem cell markers including Lgr5. These findings indicate that the miRNA cluster including miR-194 and miR-215 plays important roles in suppressing the growth and attenuating the stemness of intestinal tumor organoids.

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Anti-SIRPα antibodies as a potential new tool for cancer immunotherapy.

Yanagita T, Murata Y, Tanaka D, Motegi SI, Arai E, Daniwijaya EW, Hazama D, Washio K, Saito Y, Kotani T, Ohnishi H, Oldenborg PA, Garcia NV, Miyasaka M, Ishikawa O, Kanai Y, Komori T, Matozaki T.

JCI Insight (JCI Insight) 2 ( 1 ) e89140 2017

Accepted

© 2016 American Society for Clinical Investigation. All rights reserved. Tumor cells are thought to evade immune surveillance through interaction with immune cells. Much recent attention has focused on the modification of immune responses as a basis for new cancer treatments. SIRPα is an Ig superfamily protein that inhibits phagocytosis in macrophages upon interaction with its ligand CD47 expressed on the surface of target cells. Here, we show that SIRPα is highly expressed in human renal cell carcinoma and melanoma. Furthermore, an anti-SIRPα Ab that blocks the interaction with CD47 markedly suppressed tumor formation by renal cell carcinoma or melanoma cells in immunocompetent syngeneic mice. This inhibitory effect of the Ab appeared to be mediated by dual mechanisms: direct induction of Ab-dependent cellular phagocytosis of tumor cells by macrophages and blockade of CD47-SIRPα signaling that negatively regulates such phagocytosis. The antitumor effect of the Ab was greatly attenuated by selective depletion not only of macrophages but also of NK cells or CD8+ T cells. In addition, the anti-SIRPα Ab also enhances the inhibitory effects of Abs against CD20 and programmed cell death 1 (PD-1) on tumor formation in mice injected with SIRPα-nonexpressing tumor cells. Anti-SIRPα Abs thus warrant further study as a potential new therapy for a broad range of cancers.

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Genome-wide DNA methylation analysis during non-alcoholic steatohepatitis-related multistage hepatocarcinogenesis: comparison with hepatitis virus-related carcinogenesis.

Kuramoto J, Arai E, Tian Y, Funahashi N, Hiramoto M, Nammo T, Nozaki Y, Takahashi Y, Ito N, Shibuya A, Ojima H, Sukeda A, Seki Y, Kasama K, Yasuda K, Kanai Y.

Carcinogenesis (OXFORD UNIV PRESS) 38 ( 13 ) :261 - 270 2017

Accepted, ISSN 0008-5472

The aim of this study was to clarify the significance of DNA methylation alterations during non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis. Single-CpG-resolution genome-wide DNA methylation analysis was performed on 264 liver tissue samples using the Illumina Infinium HumanMethylation450 BeadChip. After Bonferroni correction, 3331 probes showed significant DNA methylation alterations in 113 samples of non-cancerous liver tissue showing NASH (NASH-N) as compared with 55 samples of normal liver tissue (NLT). Principal component analysis using the 3331 probes revealed distinct DNA methylation profiles of NASH-N samples that were different from those of NLT samples and 37 samples of non-cancerous liver tissue showing chronic hepatitis or cirrhosis associated with hepatitis B virus (HBV) or hepatitis C virus (HCV) infection (viral-N). Receiver operating characteristic curve analysis identified 194 probes that were able to discriminate NASH-N samples from viral-N samples with area under the curve values of more than 0.95. JonckheereTerptsra trend test revealed that DNA methylation alterations in NASH-N samples from patients without hepatocellular carcinoma (HCC) were inherited by or strengthened in NASH-N samples from patients with HCC, and then inherited by or further strengthened in 22 samples of NASH-related HCC (NASH-T) themselves. NASH-and NASH-related HCC-specific DNA methylation alterations, which were not evident in viral-N samples and 37 samples of HCC associated with HBV or HCV infection, were observed in tumor-related genes, such as WHSC1, and were frequently associated with mRNA expression abnormalities. These data suggested that NASH-specific DNA methylation alterations may participate in NASH-related multistage hepatocarcinogenesis.

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DNA and Histone Methylation in Liver Cancer

Arai E., Yotani T., Kanai Y.

Cancer Drug Discovery and Development (Cancer Drug Discovery and Development) 0 ( 9783319597843 ) 437 - 460 2017

ISSN 21969906

© 2017, Springer International Publishing AG. Epigenetic alterations, such as alterations of histone modification and DNA methylation, occur in a genome-wide manner under precancerous conditions resulting from hepatitis B virus (HBV) or hepatitis C virus (HCV) infection followed by chronic hepatitis and cirrhosis, or aberrant lipogenesis and abnormal metabolism of reactive oxygen species that characterize the pathophysiology of non-alcoholic steatohepatitis (NASH). Once DNA methylation alterations occur at the precancerous stage, they are stably preserved on DNA double strands through methylation maintenance by DNA methyltransferase 1 (DNMT1). DNA methylation alterations associated with abnormalities of DNA methyltransferase, such as overexpression of DNMT1 and splicing alterations of DNMT3B, participate in multistage hepatocarcinogenesis from the precancerous stage to the malignant progression stage and are correlated with aggressiveness of hepatocellular carcinomas (HCCs) and poorer outcome of affected patients. A number of tumor-related genes, such as ATK3, APC, BMP4, CCL20, CDH1, CDKN2A, CDKN2B, CSPG2, DAB2IP, DCC, DLC1, DPT, DPYSL3, EMILIN2, FZD7, GRASP, GSTP1, HIST1H4F, IGFALS, MGMT, MZB1, NAT2, NEFH, NFATC1, PAX4, PDSS2, PER3, PROZ, PYCARD, RASSF1A, SPDY1, RUNX3, SCGB1D1, SFN, SMPD3, SOCS1, TIMP3, TLX3, TM6SF1, TRIM33, TRIM58, WFDC6, WNK2 and ZFP41, are known to be silenced by DNA hypermethylation in human HCCs. It is believed that DNA methylation alterations could be excellent biomarkers for carcinogenetic risk estimation and prognostication. To facilitate clinical application of DNA methylation diagnosis, a scaled-down device that allows quick and accurate analysis, even in small hospitals and clinics, is now being developed. One therapeutic strategy against HCC proliferation could involve a combination of epigenetic modifiers, such as a DNA methylation inhibitor, a histone deacetylase inhibitor and an S-adenosylhomocysteine hydrolase inhibitor, to sensitize cancer cells to conventional chemotherapies, in addition to eradication of hepatitis viruses for personalized and/or pre-emptive medical care.

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発育におけるHYBID(HYaluronan-Binding protein Involved in hyaluronan Depolymerization)の機能解析

下田将之, 吉田浩之, 水野早希子, 弘實透, 堀内圭輔, 吉野雄太, 原英彰, 石島旨章, 井上紳太郎, 金井弥栄, 岡田保典

日本創傷治癒学会プログラム・抄録集 ((一社)日本創傷治癒学会) 46回 161 - 161 2016.12

Accepted

早期肺腺癌におけるEGFR遺伝子変異と腫瘍間質構成細胞変化の検討

志満敏行, 重信敬夫, 下田将之, 大塚崇, 淺村尚生, 金井弥栄

肺癌 ((NPO)日本肺癌学会) 56 ( 6 ) 583 - 583 2016.11

ISSN 0386-9628

Molecular Pathology and Novel Clinical Therapy for Uterine Leiomyosarcoma

Hayashi, Takuma, Kawano, Miki, Ichimura, Tomoyuki, Ida, Koichi, Ando, Hirofumi, Zharhary, Dorit, Kanai, Yae, Aburatani, Hiroyuki, Tonegawa, Susumu, Shiozawa, Tanri, Yaegashi, Nobuo, Konishi, Ikuo

ANTICANCER RESEARCH (INT INST ANTICANCER RESEARCH) 36 ( 10 ) 4997 - 5007 2016.10

Research paper (scientific journal), ISSN 0250-7005

Patients with uterine leiomyosarcoma (LMS) typically present with vaginal bleeding, pain, and a pelvic mass, with atypical presentations of hypercalcemia and eosinophilia also being reported. Radiographic evaluation with combined positron-emission tomography/computed tomography may assist in diagnosis and surveillance in women with uterine LMS; these are commonly used with stage and tumour grade as prognostic indicators and a recently developed risk-assessment index to predict disease-specific survival. Recent studies have shown that the addition of adjuvant therapy after surgical management does not seem to improve survival, and ovarian preservation does not appear to negatively impact outcome. Experimentally, it is noteworthy that proteasome subunit beta 9 (PSMB9)/beta 1i-deficient mice exhibit spontaneous development of uterine LMS, with a disease prevalence of similar to 37% by 12 months of age. Furthermore, a recent report showed the loss of ability to induce PSMB9/beta 1i expression, that is up-regulated by interferon-gamma (IFN gamma), in human uterine LMS tissues. Here, we reviewed human uterine LMS for genetic mutations in the IFN gamma signal cascade, and found serious mutations in three genes, Janus activated kinase 1 (JAK1), signal transducer and activator of transcription 1 (STAT1) and PSMB9/beta 1i promoter regions. Moreover, molecular experiments demonstrated differential expression of cyclin E and P27/KIP1, that regulate cell-cycle G1 arrest via PSMB9/beta 1i expression. The discovery of this mutational activation of a key cell-signalling pathway may provide new targets for diagnostic approaches and therapeutic intervention.

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【消化管II-下部消化管-】知っておきたい特殊な大腸腫瘍 UC関連dysplasia

下田, 将之, 岩男, 泰, 三上, 修治, 亀山, 香織, 金井, 弥栄

病理と臨床 34 ( 10 ) 1096 - 1101 2016.10

ISSN 0287-3745

Epigenome landscape of human normal purified hepatocytes: analysis by the International Human Epigenome Consortium (IHEC)

Eri Arai, Fumihito Miura, Yasushi Totoki, Satoshi Yamashita, Ying Tian, Masahiro Gotoh, Hidenori Ojima, Hiroyuki Nakagawa, Yoriko Takahashi, Hiromi Nakamura, Natsuko Hama, Mamoru Kato, Hiroshi Kimura, Yutaka Suzuki, Takashi Ito, Tatsuhiro Shibata, Yae Kanai

CANCER RESEARCH (AMER ASSOC CANCER RESEARCH) 76 2016.07

Accepted, ISSN 0008-5472

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ヒト正常肝細胞のエピゲノムプロファイル標準エピゲノムプロファイルと個体差

新井恵吏, 三浦史仁, 山下聡, 田迎, 後藤政広, 尾島英知, 鈴木穣, 伊藤隆司, 柴田龍弘, 金井弥栄

日本病理学会会誌 ((一社)日本病理学会) 105 ( 1 ) 361 - 361 2016.04

ISSN 0300-9181

Macroscopic features predict outcome in patients with pancreatic ductal adenocarcinoma.

Hori S, Shimada K, Ino Y, Oguro S, Esaki M, Nara S, Kishi Y, Kosuge T, Hattori Y, Sukeda A, Kitagawa Y, Kanai Y, Hiraoka N.

Virchows Arch (SPRINGER) 469 ( 6 ) 621 - 634 2016

Accepted, ISSN 09456317

As macroscopic appearance represents tumor microenvironment, it may also reflect the biological and clinicopathological characteristics of a cancer. The aim of the study was to evaluate the clinicopathological significance of the gross appearance of pancreatic ductal adenocarcinoma (PDA). We investigated fresh macroscopic features in 352 cases of PDA and their clinicopathological significance. Three unique gross features were found: a honeycomb-like appearance (diffusely distributed microcysts and interstitial fibrotic thickening), macroscopic necrosis, and a tube/branching structure (apparent small cylindrical or linear structure). A honeycomb-like appearance was present in 24 cases (6.8 %) and significantly associated with low serum CA19-9 level and well-differentiated adenocarcinoma. Macroscopic necrosis was present in 235 cases (66.8 %) and significantly correlated with tumor size, nodal metastasis, nerve plexus invasion, no adjuvant chemotherapy, and distant recurrence. The presence of macroscopic necrosis was significantly associated with shorter disease-specific survival (DSS) and disease-free survival (DFS). The presence of larger areas of necrosis (ae2 mm) was closely associated with shorter survival. A tube/branching structure was found in 179 cases (50.9 %), which was correlated with larger tumor size and no adjuvant chemotherapy and macroscopic necrosis. The presence of a tube/branching structure was significantly associated with shorter DSS and DFS. Multivariate survival analyses showed that the presence of tube/branching structures was an independent negative prognostic factor in patients having PDA. We suggest that the gross appearance of PDA reflects clinicopathological characteristics and may be useful in predicting prognosis.

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Lipidomic Signatures and Associated Transcriptomic Profiles of Clear Cell Renal Cell Carcinoma.

Saito K, Arai E, Maekawa K, Ishikawa M, Fujimoto H, Taguchi R, Matsumoto K, Kanai Y, Saito Y.

Sci Rep (NATURE PUBLISHING GROUP) 6 28932 2016

Accepted, ISSN 2045-2322

Renal cell carcinoma (RCC) is the most common histological type of adult kidney cancer. In this study, we obtained lipidomic profiles of clear cell RCC (ccRCC), a major RCC subtype, by performing a lipidomic analysis of specimens of cancerous tissue and the surrounding normal renal cortex obtained from the same patients (N = 49). We also compared the lipidomic profiles with the lipogenic transcriptome of specimens of cancerous tissue and the surrounding normal renal cortex for an additional set of patient samples (N = 95). Overall, we detected 326 lipids, including phospholipids, sphingolipids, neutral lipids, and eicosanoids. The levels of more than 70% of the detected lipids were significantly different (P < 0.01, corrected by the false discovery rate). The cancerous tissue was distinguished by higher levels of ether-type phospholipids, cholesterol esters, and triacylglycerols, as well as by lower levels of phospholipids (except for phosphatidylcholines) and polyunsaturated fatty acids. Characteristic changes in the levels of mRNAs and metabolites suggested that the phosphatidylethanolamine (PE) synthesis pathway is suppressed in ccRCC and associated with cell proliferation. The present study represents the lipidomic profiles of ccRCC, which provides novel information about the metabolic changes in renal cancerous tissue and RCC pathophysiology.

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Conditional abrogation of Atm in osteoclasts extends osteoclast lifespan and results in reduced bone mass.

Hirozane T, Tohmonda T, Yoda M, Shimoda M, Kanai Y, Matsumoto M, Morioka H, Nakamura M, Horiuchi K.

Sci Rep (NATURE PUBLISHING GROUP) 6 34426 2016

Accepted, ISSN 2045-2322

Ataxia-telangiectasia mutated (ATM) kinase is a central component involved in the signal transduction of the DNA damage response (DDR) and thus plays a critical role in the maintenance of genomic integrity. Although the primary functions of ATM are associated with the DDR, emerging data suggest that ATM has many additional roles that are not directly related to the DDR, including the regulation of oxidative stress signaling, insulin sensitivity, mitochondrial homeostasis, and lymphocyte development. Patients and mice lacking ATM exhibit growth retardation and lower bone mass; however, the mechanisms underlying the skeletal defects are not fully understood. In the present study, we generated mutant mice in which ATM is specifically inactivated in osteoclasts. The mutant mice did not exhibit apparent developmental defects but showed reduced bone mass due to increased osteoclastic bone resorption. Osteoclasts lacking ATM were more resistant to apoptosis and showed a prolonged lifespan compared to the controls. Notably, the inactivation of ATM in osteoclasts resulted in enhanced NF-kappa B signaling and an increase in the expression of NF-kappa B-targeted genes. The present study reveals a novel function for ATM in regulating bone metabolism by suppressing the lifespan of osteoclasts and osteoclast-mediated bone resorption.

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SMYD2 overexpression is associated with tumor cell proliferation and a worse outcome in human papillomavirus-unrelated nonmultiple head and neck carcinomas.

Ohtomo-Oda R, Komatsu S, Mori T, Sekine S, Hirajima S, Yoshimoto S, Kanai Y, Otsuji E, Ikeda E, Tsuda H.

Hum Pathol (W B SAUNDERS CO-ELSEVIER INC) 49 145 - 155 2016

Accepted, ISSN 00468177

Human papillomavirus (HPV) unrelated head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease, and there are no suitable prognostic or predictive markers. The SET and MYND domain-containing protein 2 (SMYD2), a lysine methyltransferase for histone H31(36 and p53K370, that regulates transcription was previously found to be a cancer-promoting gene in esophageal squamous cell carcinoma. In this study, we investigated whether SMYD2 is a possible oncogene and a prognostic indicator in HPV-unrelated, multiple and nonmultiple HNSCC. Among 197 HPV-unrelated HNSCC cases, over expression of SMYD2 protein was detected in 75 (60%) of 126 nonmultiple cases and 51 (70%) of 71 multiple cases. In nonmultiple cases, patients with SMYD2-overexpressing tumors had a worse overall survival rate than did those with nonexpressing tumors (P = .017, log-rank test), and SMYD2 positivity was independently associated with overall survival in the multivariate analysis (P = .003). In both nonmultiple and multiple groups, the combination of SMYD2 and p53 immunopositivity was a significant prognostic indicator (P = .027 and.015). In 5 HNSCC cell lines, overexpression of SMYD2 messenger RNA and protein was observed, but there was no notable amplification at 1q32-41.1. The proliferation of UM-SCC-17B HPV-unrelated HNSCC cell line was inhibited by knockdown of SMYD2 gene expression. These findings suggest that SMYD2 plays a role in tumor progression and might be a useful prognosticator in HPV-unrelated, nonmultiple HNSCC. (C) 2015 Elsevier Inc. All rights reserved.

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Genomic landscape of experimental bladder cancer in rodents and its application to human bladder cancer: Gene amplification and potential overexpression of Cyp2a5/CYP2A6 are associated with the invasive phenotype.

Kanemoto K, Fukuta K, Kawai N, Tozawa K, Ochiai M, Okamoto K, Ohnami S, Sakamoto H, Yoshida T, Kanai Y, Katoh M, Yasui T, Kohri K, Kakizoe T, Nakagama H.

PLoS One (PUBLIC LIBRARY SCIENCE) 11 ( 11 ) e0167374 2016

Accepted, ISSN 1932-6203

Non-muscle invasive (superficial) bladder cancer is a low-grade malignancy with good prognosis, while muscle invasive (invasive) bladder cancer is a high-grade malignancy with poor prognosis. N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) induces superficial bladder cancers with papillary morphology in rats and invasive bladder cancers with infiltrating phenotype in mice. In this study, we analyzed genomic landscapes of rodent BBN-induced bladder cancers using array-based comparative genomic hybridization (array CGH). While no significant copy number alterations were detected in superficial bladder tumors in rats, copy number gains in chromosomal regions 2D-E1, 7qA3, 9F2, and 11C-D were detected in invasive bladder tumors in mice. Amplification of representative genes located on 2D-E1 and 7qA3 chromosomal regions was confirmed by quantitative PCR. Cyp2a22 and Cyp2a5 genes but not Cyp2g1, Cyp2a12, and Rab4b genes on mouse chromosome 7qA3 were amplified in invasive bladder cancers. Although the human ortholog gene of Cyp2a22 has not been confirmed, the mouse Cyp2a5 gene is the ortholog of the human CYP2A6 gene located in chromosomal region 19q13.2, and CYP2A6 was identified by database search as one of the closest human homolog to mouse Cyp2a22. Considering a possibility that this region may be related to mouse 7qA3, we analyzed CYP2A6 copy number and expression in human bladder cancer using cell lines and resected tumor specimens. Although only one of eight cell lines showed more than one copy increase of the CYP2A6 gene, CYP2A6 amplification was detected in six out of 18 primary bladder tumors where it was associated with the invasive phenotype. Immunohistochemical analyses of 118 primary bladder tumors revealed that CYP2A6 protein expression was also higher in invasive tumors, especially in those of the scattered type. Together, these findings indicate that the amplification and overexpression of the CYP2A6 gene are characteristic of human bladder cancers with increased malignancy and that CYP2A6 can be a candidate prognostic biomarker in this type of cancer.

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Mieap-regulated mitochondrial quality control is frequently inactivated in human colorectal cancer.

Kamino H, Nakamura Y, Tsuneki M, Sano H, Miyamoto Y, Kitamura N, Futamura M, Kanai Y, Taniguchi H, Shida D, Kanemitsu Y, Moriya Y, Yoshida K, Arakawa H.

Oncogenesis (NATURE PUBLISHING GROUP) 4 e181 2016

Accepted, ISSN 2157-9024

Mieap, a p53-inducible protein, controls mitochondrial quality by repairing or eliminating unhealthy mitochondria. BNIP3 and NIX are critical mediators for the Mieap-regulated mitochondrial quality control. Mieap suppresses murine intestinal tumor via its mitochondrial quality control function. To explore the role of the Mieap-regulated mitochondria quality control function in colorectal cancer patients, we examined the statuses of p53, Mieap, BNIP3 and NIX in 57 primary colorectal cancer tissues. Promoter methylation of the Mieap and BNIP3 genes was found in 9% and 47% of colorectal cancer cases, respectively, whereas p53 mutation was found in more than 50% of colorectal cancer tissues lacking methylation of the Mieap and BNIP3 promoters, implying that the p53/Mieap/BNIP3-regulated mitochondria quality control pathway is inactivated in more than 70% of colorectal cancer patients. In LS174T colorectal cancer cells, hypoxia activated the Mieap-regulated mitochondria quality control function. Knockdown of p53, Mieap or BNIP3 in LS174T cells severely impaired the hypoxia-activated function, leading to the accumulation of unhealthy mitochondria and increase of mitochondrial reactive oxygen species generation. The mitochondrial reactive oxygen species generated by unhealthy mitochondria in the p53/Mieap/BNIP3-deficient cells remarkably enhanced cancer cell migration and invasion under hypoxic condition. These results suggest that the Mieap-regulated mitochondria quality control has a critical role in colorectal cancer suppression in the in vivo hypoxic tumor microenvironment.

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自閉症スペクトラムのエピゲノム異常（和訳）

下田将之, 金井弥栄

実験医学 34 1662 - 1668 2016

TERT promoter mutations are frequent and show association with MED12 mutations in phyllodes tumors of the breast

Yoshida, Masayuki, Ogawa, Reiko, Yoshida, Hiroshi, Maeshima, Akiko, Kanai, Yae, Kinoshita, Takayuki, Hiraoka, Nobuyoshi, Sekine, Shigeki

BRITISH JOURNAL OF CANCER (NATURE PUBLISHING GROUP) 113 ( 8 ) 1244 - 1248 2015.10

Research paper (scientific journal), ISSN 0007-0920

Background: Phyllodes tumors are rare fibroepithelial neoplasms of the breast, which carry the potential risk of local recurrence and metastasis. Phyllodes tumors share several histological features with fibroadenomas, and no widely accepted markers for distinguishing these lesions have been identified.
Methods: We analyzed molecular abnormalities related to telomere elongation in tumors, including TERT promoter mutations, as well as loss of expression of ATRX and DAXX, in a total of 104 phyllodes tumors and fibroadenomas.
Results: Sequencing analyses showed that TERT promoter mutations were frequent in phyllodes tumors (30/46, 65%), but rare in fibroadenomas (4/58, 7%). Among phyllodes tumors, the mutations were more frequent in borderline tumors (13/15, 87%), but were also common in benign (9/18, 50%) and malignant tumors (8/13, 62%). Remarkably, all but one TERT promoter-mutated tumor also contained MED12 mutations, indicating that these mutations are strongly associated (P = 8.4 x 10(-6)). Expression of ATRX and DAXX, as evaluated by immunohistochemistry, was retained in all tumors.
Conclusions: Our observations suggest a critical role of TERT promoter mutations, in cooperation with MED12 mutations, in the development of phyllodes tumors. Because TERT promoter mutations are rare among fibroadenomas, their detection may be of potential use in discriminating between phyllodes tumors and fibroadenomas.

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Prognostic implication of PTPRH hypomethylation in non-small cell lung cancer

Sato, Taicashi, Soejima, Kenzo, Arai, Eri, Hamamoto, Junko, Yasuda, Hiroyuki, Arai, Daisuke, Ishioka, Kota, Ohgino, Keiko, Naoki, Katsuhiko, Kohno, Takashi, Tsuta, Koji, Watanabe, Shun-Ichi, Kanai, Yae, Betsuyaku, Tomoko

ONCOLOGY REPORTS (SPANDIDOS PUBL LTD) 34 ( 3 ) 1137 - 1145 2015.09

Research paper (scientific journal), ISSN 1021-335X

PTPRH is a receptor-type protein tyrosine phosphatase thought to be a potential regulator of tumorigenesis. The aim of the present study was to clarify the significance of PTPRH expression and its regulation by DNA methylation in non-small cell lung cancer (NSCLC), especially in lung adenocarcinoma (LADC). PTPRH mRNA expression was examined in 89 NSCLC and corresponding non-cancerous tissues. The correlation between DNA methylation and PTPRH gene expression was investigated in another cohort that consisted of 145 patients with LADC, a major NSCLC subtype. Gene regulation by DNA methylation was assessed using a DNA methylation inhibitor. PTPRH mRNA expression was significantly upregulated in NSCLC. PTPRH DNA methylation was reduced in LADC samples and inversely correlated with mRNA expression. 5-Aza-2'-deoxycytidine treatment of lung cancer cell lines with low PTPRH expression, restored mRNA PTPRH expression levels. Furthermore, low PTPRH methylation was associated with shorter recurrence-free survival (P=1.64x10(-4)) and overall survival (P=5.54x10(-5)). Multivariate analysis revealed that PTPRH DNA methylation was an independent prognostic factor (P=6.88x10(-3)). It was confirmed that PTPRH is overexpressed in NSCLC. Furthermore, we determined that PTPRH is epigenetically regulated by DNA hypomethylation, with prognostic implications for LADC.

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Heat Shock Protein 90 Is a Potential Therapeutic Target in Cholangiocarcinoma

Shirota, Tomoki, Ojima, Hidenori, Hiraoka, Nobuyoshi, Shimada, Kazuaki, Rokutan, Hirofumi, Arai, Yasuhito, Kanai, Yae, Miyagawa, Shinichi, Shibata, Tatsuhiro

MOLECULAR CANCER THERAPEUTICS (AMER ASSOC CANCER RESEARCH) 14 ( 9 ) 1985 - 1993 2015.09

Research paper (scientific journal), ISSN 1535-7163

Cholangiocarcinoma is an aggressive malignancy with a poor prognosis, with no effective therapy other than surgical resection. Heat shock protein 90 (HSP90) is a key component of a multi-chaperone complex involved in the posttranslational folding of a number of client proteins, many of which play essential roles in tumorigenesis. Here, we attempted to clarify its prognostic significance and potential utility as a therapeutic target in cholangiocarcinoma. Immunohistochemical expression of HSP90 was assessed retrospectively in 399 cholangiocarcinoma cases and 17 human cholangiocarcinoma cell lines, along with the effect of a small-molecule HSP90 inhibitor (NVP-AUY922) on cholangiocarcinoma tumor growth and angiogenesis in human cholangiocarcinoma cell lines and xenografts. The positivity of HSP90 was 44.6% in intrahepatic cholangiocarcinoma (IHCC) and 32.8% in extrahepatic cholangiocarcinoma (EHCC), respectively. HSP90 expression was significantly associated with the 5-year survival rate for IHCC (P < 0.001) and EHCC (P < 0.001). HSP90 inhibition showed potent antiproliferative activity and reduced growth-associated signaling in human cholangiocarcinoma cells in vitro. Furthermore, treatment of cholangiocarcinoma xenograft-bearingmice with NVP-AUY922 significantly inhibited growth at doses far below the maximum-tolerated dose. HSP90 overexpression is a prognostic marker for cholangiocarcinoma. HSP90-targeted therapy may be an option for a subset of cholangiocarcinoma. (C) 2015 AACR.

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An Integrated Prognostic Classifier for Stage I Lung Adenocarcinoma Based on mRNA, microRNA, and DNA Methylation Biomarkers

Robles, Ana I., Arai, Eri, Mathe, Ewy A., Okayama, Hirokazu, Schetter, Aaron J., Brown, Derek, Petersen, David, Bowman, Elise D., Noro, Rintaro, Welsh, Judith A., Edelman, Daniel C., Stevenson, Holly S., Wang, Yonghong, Tsuchiya, Naoto, Kohno, Takashi, Skaug, Vidar, Mollerup, Steen, Haugen, Aage, Meltzer, Paul S., Yokota, Jun, Kanai, Yae, Harris, Curtis C.

JOURNAL OF THORACIC ONCOLOGY (LIPPINCOTT WILLIAMS & WILKINS) 10 ( 7 ) 1037 - 1048 2015.07

Research paper (scientific journal), ISSN 1556-0864

Introduction: Up to 30% stage I lung cancer patients suffer recurrence within 5 years of curative surgery. We sought to improve existing protein-coding gene and microRNA expression prognostic classifiers by incorporating epigenetic biomarkers.
Methods: Genome-wide screening of DNA methylation and pyrosequencing analysis of HOXA9 promoter methylation were performed in two independently collected cohorts of stage I lung adenocarcinoma. The prognostic value of HOXA9 promoter methylation alone and in combination with mRNA and miRNA biomarkers was assessed by Cox regression and Kaplan-Meier survival analysis in both cohorts.
Results: Promoters of genes marked by polycomb in embryonic stem cells were methylated de novo in tumors and identified patients with poor prognosis. The HOXA9 locus was methylated de novo in stage I tumors (p < 0.0005). High HOXA9 promoter methylation was associated with worse cancer-specific survival (hazard ratio [HR], 2.6; p = 0.02) and recurrence-free survival (HR, 3.0; p = 0.01), and identified high-risk patients in stratified analysis of stages IA and IB. Four protein-coding gene (XPO1, BRCA1, HIF1, and DLC1), miR-21 expression, and HOXA9 promoter methylation were each independently associated with outcome (HR, 2.8; p = 0.002; HR, 2.3; p = 0.01; and HR, 2.4; p = 0.005, respectively), and when combined, identified high-risk, therapy naive, stage I patients (HR, 10.2; p = 3x10(-5)). All associations were confirmed in two independently collected cohorts.
Conclusion: A prognostic classifier comprising three types of genomic and epigenomic data may help guide the postoperative management of stage I lung cancer patients at high risk of recurrence.

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Epigenetic clustering of gastric carcinomas based on DNA methylation profiles at the precancerous stage: its correlation with tumor aggressiveness and patient outcome

Yamanoi, Kazuhiro, Arai, Eri, Tian, Ying, Takahashi, Yoriko, Miyata, Sayaka, Sasaki, Hiroki, Chiwaki, Fumiko, Ichikawa, Hitoshi, Sakamoto, Hiromi, Kushima, Ryoji, Katai, Hitoshi, Yoshida, Teruhiko, Sakamoto, Michiie, Kanai, Yae

CARCINOGENESIS (OXFORD UNIV PRESS) 36 ( 5 ) 509 - 520 2015.05

Research paper (scientific journal), ISSN 0143-3334

Single-CpG resolution genome-wide DNA methylation analysis indicated that distinct DNA methylation profiles are established during field cancerization in gastric mucosae, and such profiles at the precancerous stage are inherited by gastric cancers, thus determining tumor aggressiveness and patient outcome.The aim of this study was to clarify the significance of DNA methylation alterations during gastric carcinogenesis. Single-CpG resolution genome-wide DNA methylation analysis using the Infinium assay was performed on 109 samples of non-cancerous gastric mucosa (N) and 105 samples of tumorous tissue (T). DNA methylation alterations in T samples relative to N samples were evident for 3861 probes. Since N can be at the precancerous stage according to the field cancerization concept, unsupervised hierarchical clustering based on DNA methylation levels was performed on N samples (beta(N)) using the 3861 probes. This divided the 109 patients into three clusters: A (n = 20), B1 (n = 20), and B2 (n = 69). Gastric carcinomas belonging to Cluster B1 showed tumor aggressiveness more frequently than those belonging to Clusters A and B2. The recurrence-free and overall survival rates of patients in Cluster B1 were lower than those of patients in Clusters A and B2. Sixty hallmark genes for which beta(N) characterized the epigenetic clustering were identified. We then focused on DNA methylation levels in T samples (beta(T)) of the 60 hallmark genes. In 48 of them, including the ADAM23, OLFM4, AMER2, GPSM1, CCL28, DTX1 and COL23A1 genes, beta(T) was again significantly correlated with tumor aggressiveness, and the recurrence-free and/or overall survival rates. Multivariate analyses revealed that beta(T) was a significant prognostic factor, being independent of clinicopathological parameters. These data indicate that DNA methylation profiles at the precancerous stage may be inherited by gastric carcinomas themselves, thus determining tumor aggressiveness and patient outcome.

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Tumour multifocality and grade predict intravesical recurrence after nephroureterectomy in patients with upper urinary tract urothelial carcinoma without a history of bladder cancer

Narukawa, Tsukasa, Hara, Tomohiko, Arai, Eri, Komiyama, Motokiyo, Kawahara, Takashi, Kanai, Yae, Fujimoto, Hiroyuki

JAPANESE JOURNAL OF CLINICAL ONCOLOGY (OXFORD UNIV PRESS) 45 ( 5 ) 488 - 493 2015.05

Research paper (scientific journal), ISSN 0368-2811

Objective: Patients with upper urinary tract urothelial carcinoma (UUT-UC) without a history of bladder cancer have a different natural history of intravesical recurrence after nephroureterectomy compared with those with a history of bladder cancer. The aim of this study was to identify predictive factors for post-operative intravesical recurrence in patients with non-metastatic upper urinary tract-localized urothelial carcinoma without a history of bladder cancer and who were not taking medication during the perioperative period.
Methods: This retrospective study included 133 patients who were treated between 1995 and 2012. Univariate and multivariate analyses were used to evaluate the clinical and pathological factors associated with the cumulative incidence of bladder cancer.
Results: Of the 133 patients, 51 (38.3%) developed intravesical recurrence during a median follow-up of 71 months (range, 0.8-210.8). In the multivariate analysis, multifocality (P = 0.03) and high tumour grade (P = 0.007) were significantly associated with the cumulative incidence of bladder cancer. We constructed a prediction classification model on the basis of the total number of risk factors. The 2-year cumulative incidence rates were 5.6, 34.8 and 50.0% in individuals with no, one and two risk factors, respectively. There was a significant difference between patients with no risk factors and those with two risk factors (P = 0.01).
Conclusions: Although this retrospective study had several limitations, tumour multifocality and tumour grade were found to be potential risk factors for intravesical recurrence in our cases.

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Activating GNAS and KRAS mutations in gastric foveolar metaplasia, gastric heterotopia, and adenocarcinoma of the duodenum

A. Matsubara, R. Ogawa, H. Suzuki, I. Oda, H. Taniguchi, Y. Kanai, R. Kushima, S. S Sekine

BRITISH JOURNAL OF CANCER (NATURE PUBLISHING GROUP) 112 ( 8 ) 1398 - 1404 2015.04

Accepted, ISSN 00070920

Background: Heterotopic gastric-type epithelium, including gastric foveolar metaplasia (GFM) and gastric heterotopia (GH), is a common finding in duodenal biopsy specimens; however, there is still controversy regarding their histogenetic backgrounds.
Methods: We analysed a total of 177 duodenal lesions, including 66 GFM lesions, 81 GH lesions, and 30 adenocarcinomas, for the presence of GNAS, KRAS, and BRAF mutations.
Results: Activating GNAS mutations were identified in 27 GFM lesions (41%) and 23 GH lesions (28%). The KRAS mutations were found in 17 GFM lesions (26%) and 2 GH lesions (2%). A BRAF mutation was found in only one GFM lesion (2%). These mutations were absent in all 32 normal duodenal mucosa specimens that were examined, suggesting a somatic nature. Among the GFM lesions, GNAS mutations were more common in lesions without active inflammation. Analyses of adenocarcinomas identified GNAS and KRAS mutations in 5 (17%) and 11 lesions (37%), respectively. Immunohistochemically, all the GNAS-mutated adenocarcinomas diffusely expressed MUC5AC, indicating gastric epithelial differentiation.
Conclusions: A significant proportion of GFM and GH harbours GNAS and/or KRAS mutations. The common presence of these mutations in duodenal adenoma and adenocarcinoma with a gastric epithelial phenotype implies that GFM and GH might be precursors of these tumours.

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Lysine Demethylase LSD1 Coordinates Glycolytic and Mitochondrial Metabolism in Hepatocellular Carcinoma Cells

Sakamoto, Akihisa, Hino, Shinjiro, Nagaoka, Katsuya, Anan, Kotaro, Takase, Ryuta, Matsumori, Haruka, Ojima, Hidenori, Kanai, Yae, Arita, Kazunori, Nakao, Mitsuyoshi

CANCER RESEARCH (AMER ASSOC CANCER RESEARCH) 75 ( 7 ) 1445 - 1456 2015.04

Research paper (scientific journal), ISSN 0008-5472

The hallmark of most cancer cells is the metabolic shift from mitochondrial to glycolytic metabolism for adapting to the surrounding environment. Although epigenetic modification is intimately linked to cancer, the molecular mechanism, by which epigenetic factors regulate cancer metabolism, is poorly understood. Here, we show that lysine-specific demethylase-1 (LSD1, KDM1A) has an essential role in maintaining the metabolic shift in human hepatocellular carcinoma cells. Inhibition of LSD1 reduced glucose uptake and glycolytic activity, with a concurrent activation of mitochondrial respiration. These metabolic changes coexisted with the inactivation of the hypoxia-inducible factor HIF1 alpha, resulting in a decreased expression of GLUT1 and glycolytic enzymes. In contrast, during LSD1 inhibition, a set of mitochondrial metabolism genes was activated with the concomitant increase of methylated histone H3 at lysine 4 in the promoter regions. Consistently, both LSD1 and GLUT1 were significantly overexpressed in carcinoma tissues. These findings demonstrate the epigenetic plasticity of cancer cell metabolism, which involves an LSD1-mediated mechanism. (C)2015 AACR.

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The excision repair cross-complementation group 1

Sakamoto Y, Ojima H, Morizane C, Kanai Y, Okusaka T

Nihon rinsho. Japanese journal of clinical medicine (Nihon rinsho. Japanese journal of clinical medicine) 73 485 - 490 2015.03

ISSN 00471852

家族性大腸腺腫症に伴う幽門腺腺腫と散発性幽門腺腺腫は共通の遺伝子変異を有する

橋本大輝, 小川玲子, 松原亜季子, 谷口浩和, 菅野康吉, 牛尼美年子, 吉田輝彦, 金井弥栄, 関根茂樹

日本病理学会会誌 ((一社)日本病理学会) 104 ( 1 ) 305 - 305 2015.03

ISSN 0300-9181

Long-term exposure to gefitinib induces acquired resistance through DNA methylation changes in the EGFR-mutant PC9 lung cancer cell line

Terai, Hideki, Soejima, Kenzo, Yasuda, Hiroyuki, Sato, Takashi, Naoki, Katsuhiko, Ikemura, Shinnosuke, Arai, Daisuke, Ohgino, Keiko, Ishioka, Kota, Hamamoto, Junko, Kanai, Yae, Betsuyaku, Tomoko

INTERNATIONAL JOURNAL OF ONCOLOGY (SPANDIDOS PUBL LTD) 46 ( 1 ) 430 - 436 2015.01

Research paper (scientific journal), ISSN 1019-6439

This study was designed to identify epigenetically regulated genes and to clarify the contribution of epigenetic alteration to acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). We established a gefitinib-resistant lung cancer cell line, PC9, which was originally gefitinib-sensitive, by serial long-term exposure to gefitinib. RNA and DNA were collected from both gefitinib-sensitive and -resistant PC9 cells, and comprehensive DNA methylation and mRNA expression analyses were performed using Infinium HumanMethylation27 Bead Arrays and Agilent SurePrint G3 Human Gene Expression 8x60K Array, respectively. DNA methylation was increased in 640 genes in gefitinib-resistant cells compared to parental cells. Among them, we selected 29 candidate genes that presented a decrease in mRNA expression in resistant PC9. We further studied four of the selected genes (C10orf116, IGFBP3, KL, and S100P) and found that KL or S100P silencing by siRNA induced a decrease in gefitinib sensitivity compared to that in the negative control in PC9. In conclusion, KL and S100P could be potential targets to overcome resistance to EGFR-TKIs.

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Alterations of the spindle checkpoint pathway in clinicopathologically aggressive CpG island methylator phenotype clear cell renal cell carcinomas.

Arai E, Gotoh M, Tian Y, Sakamoto H, Ono M, Matsuda A, Takahashi Y, Miyata S, Totsuka H, Chiku S, Komiyama M, Fujimoto H, Matsumoto K, Yamada T, Yoshida T, Kanai Y.

Int J Cancer (WILEY) 137 ( 11 ) 2589 - 2606 2015

Accepted, ISSN 00207136

CpG-island methylator phenotype (CIMP)-positive clear cell renal cell carcinomas (RCCs) are characterized by accumulation of DNA hypermethylation of CpG islands, clinicopathological aggressiveness and poor patient outcome. The aim of this study was to clarify the molecular pathways participating in CIMP-positive renal carcinogenesis. Genome (whole-exome and copy number), transcriptome and proteome (two-dimensional image converted analysis of liquid chromatography-mass spectrometry) analyses were performed using tissue specimens of 87 CIMP-negative and 14 CIMP-positive clear cell RCCs and corresponding specimens of non-cancerous renal cortex. Genes encoding microtubule-associated proteins, such as DNAH2, DNAH5, DNAH10, RP1 and HAUS8, showed a 10% or higher incidence of genetic aberrations (non-synonymous single-nucleotide mutations and insertions/deletions) in CIMP-positive RCCs, whereas CIMP-negative RCCs lacked distinct genetic characteristics. MetaCore pathway analysis of CIMP-positive RCCs revealed that alterations of mRNA or protein expression were significantly accumulated in six pathways, all participating in the spindle checkpoint, including the "The metaphase checkpoint (p=1.427 x 10(-6))," "Role of Anaphase Promoting Complex in cell cycle regulation (p=7.444 x 10(-6))" and " Spindle assembly and chromosome separation (p=9.260 x 10(-6))" pathways. Quantitative RT-PCR analysis revealed that mRNA expression levels for genes included in such pathways, i.e., AURKA, AURKB, BIRC5, BUB1, CDC20, NEK2 and SPC25, were significantly higher in CIMP-positive than in CIMP-negative RCCs. All CIMP-positive RCCs showed overexpression of Aurora kinases, AURKA and AURKB, and this overexpression was mainly attributable to increased copy number. These data suggest that abnormalities of the spindle checkpoint pathway participate in CIMP-positive renal carcinogenesis, and that AURKA and AURKB may be potential therapeutic targets in more aggressive CIMP-positive RCCs.

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Biological characterization of soft tissue sarcomas.

Hayashi T, Horiuchi A, Sano K, Kanai Y, Yaegashi N, Aburatani H, Konishi I.

Ann Transl Med 3 368 2015

Accepted

Frequent MED12 mutations in phyllodes tumours of the breast.

Yoshida M, Sekine S, Ogawa R, Yoshida H, Maeshima A, Kanai Y, Kinoshita T, Ochiai A.

Br J Cancer (NATURE PUBLISHING GROUP) 112: ( 10 ) 1703 - 1708 2015

Accepted, ISSN 00070920

Background: Phyllodes tumours are rare fibroepithelial tumours of the breast, that include benign, borderline, and malignant lesions. Although the molecular basis of phyllodes tumours largely remains unknown, a recent exome study identified MED12 mutations as a sole recurrent genetic alteration in fibroadenoma, a common benign fibroepithelial tumour that shares some histological features with the phyllodes tumour.
Methods: Forty-six phyllodes tumours and 58 fibroadenomas of the breast were analysed for MED12 mutations by using Sanger sequencing.
Results: MED12 mutations were identified in 37 out of the 46 phyllodes tumours (80%). The prevalence of MED12 mutations was similar among benign (15/18, 83%), borderline (12/15, 80%), and malignant tumours (10/13, 77%). MED12 mutations were also identified in 36 of the 58 fibroadenomas (62%). The mutations were frequent among intracanalicular-type (24/32, 75%) and complex-type lesions (4/6, 67%), but were significantly less common among the pericanalicular-type lesions (8/20, 40%). A microdissection-based analysis showed that MED12 mutations were confined to the stromal components in both phyllodes tumours and fibroadenomas.
Conclusions: MED12 mutations were frequent among the phyllodes tumours of the breast, regardless of the tumour grade. Phyllodes tumours and fibroadenomas share, at least in part, a common genetic background.

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Intratumoral tertiary lymphoid organ is a favourable prognosticator in patients with pancreatic cancer.

Hiraoka N, Ino Y, Yamazaki-Itoh R, Kanai Y, Kosuge T, Shimada K.

Br J Cancer (NATURE PUBLISHING GROUP) 112 ( 11 ) 1782 - 1790 2015

Accepted, ISSN 00070920

Background: Host immunity has critical roles in tumour surveillance. Tertiary lymphoid organs (TLOs) are induced in various inflamed tissues. The aim of this study was to investigate the clinicopathological and pathobiological characteristics of tumour microenvironment in pancreatic ductal carcinoma (PDC) with TLOs.
Methods: We examined 534 PDCs to investigate the clinicopathological impact of TLOs and their association with tumour-infiltrating immune cells, the cytokine milieu, and tissue characteristics.
Results: There were two different localisations of PDC-associated TLOs, intratumoral and peritumoral. A better outcome was observed in patients with intratumoral TLOs, and this was independent of other survival factors. The PDC tissues with intratumoral TLOs showed significantly higher infiltration of T and B cells and lower infiltration of immunosuppressive cells, as well as significantly higher expression of Th1-and Th17-related genes. Tertiary lymphoid organs developed with an association with arterioles, venules, and nerves. These structures were reduced in an association with cancer invasion in PDC tissues, except for those with intratumoral TLOs. The PDC tissues with intratumoral TLOs had capillaries consisting of mature endothelial cells covered by pericytes.
Conclusions: Our results suggest that the presence of intratumoral TLOs represents a microenvironment that has an active immune reaction, and shows a relatively intact vascular network retained.

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Activating GNAS and KRAS mutations in gastric foveolar metaplasia, gastric heterotopia and adenocarcinoma of the duodenum.

Matsubara A, Ogawa R, Suzuki H, Oda I, Taniguchi H, Kanai Y, Kushima R, Sekine S.

Br J Cancer 112 1398 - 1404 2015

Accepted

Familial adenomatous polyposis-associated and sporadic pyloric gland adenomas of the upper gastrointestinal tract share common genetic features.

Hashimoto T, Ogawa R, Matsubara A, Taniguchi H, Sugano K, Ushiama M, Yoshida T, Kanai Y, Sekine S.

Histopathology (WILEY-BLACKWELL) 67 ( 5 ) 689 - 698 2015

Accepted, ISSN 03090167

AimsFamilial adenomatous polyposis (FAP) is a hereditary cancer predisposition syndrome caused by a germline APC mutation. A recent study showed the enrichment of pyloric gland adenomas (PGAs) of the stomach, in addition to fundic gland polyps (FGPs) and foveolar-type adenomas (FAs), in patients with FAP. In the present study, we analysed the genetic alterations in these FAP-associated gastric lesions.
Methods and resultsMutational statuses of GNAS and KRAS, which are frequently mutated in sporadic PGAs, as well as those of APC, were examined in PGAs, FAs and FGPs in patients with FAP using Sanger sequencing. Our analysis identified GNAS mutations in five of six PGAs (83%), but in none of the three FAs or the 40 FGPs examined. KRAS mutations were identified in four PGAs (67%), one FA (33%) and one FGP (3%). Somatic truncating APC mutations were found in all PGAs (100%), two FAs (67%) and 14 FGPs (47%). We additionally analysed sporadic PGAs of the stomach and duodenum and identified truncating APC mutations in 11 of 25 lesions (44%).
ConclusionsFAP-associated and sporadic PGAs not only show similar morphologies, but also share common genetic aberrations, including mutations of GNAS, KRAS and APC.

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The importance of multifocality and tumor grade for predicting bladder cancer recurrence after nephroureterectomy in patients with non-metastatic upper urinary tract cancer without a history of bladder cancer.

Narukawa T, Hara T, Arai E, Komiyama M, Kawahara T, Kanai Y, Fujimoto H.

Int J Urol 45 488 - 493 2015

Accepted

Integrated omics analysis: development of companion biomarkers and identification of therapeutic targets

Kanai Yae, Arai Eri

Japan Journal of Molecular Tumor Marker Research (Japanese Society for Molecular Tumor Marker Research) 31 15 - 16 2015

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Comprehensive Exploration of Novel Chimeric Transcripts in Clear Cell Renal Cell Carcinomas Using Whole Transcriptome Analysis

Gotoh, Masahiro, Ichikawa, Hitoshi, Arai, Eri, Chiku, Suenori, Sakamoto, Hiromi, Fujimoto, Hiroyuki, Hiramoto, Masaki, Nammo, Takao, Yasuda, Kazuki, Yoshida, Teruhiko, Kanai, Yae

GENES CHROMOSOMES & CANCER (WILEY-BLACKWELL) 53 ( 12 ) 1018 - 1032 2014.12

Research paper (scientific journal), ISSN 1045-2257

The aim of this study was to clarify the participation of expression of chimeric transcripts in renal carcinogenesis. Whole transcriptome analysis (RNA sequencing) and exploration of candidate chimeric transcripts using the deFuse program were performed on 68 specimens of cancerous tissue (T) and 11 specimens of non-cancerous renal cortex tissue (N) obtained from 68 patients with clear cell renal cell carcinomas (RCCs) in an initial cohort. As positive controls, two RCCs associated with Xp11.2 translocation were analyzed. After verification by reverse transcription (RT)-PCR and Sanger sequencing, 26 novel chimeric transcripts were identified in 17 (25%) of the 68 clear cell RCCs. Genomic breakpoints were determined in five of the chimeric transcripts. Quantitative RT-PCR analysis revealed that the mRNA expression levels for the MMACHC, PTER, EPC2, ATXN7, FHIT, KIFAP3, CPEB1, MINPP1, TEX264, FAM107A, UPF3A, CDC16, MCCC1, CPSF3, and ASAP2 genes, being partner genes involved in the chimeric transcripts in the initial cohort, were significantly reduced in 26 T samples relative to the corresponding 26 N samples in the second cohort. Moreover, the mRNA expression levels for the above partner genes in T samples were significantly correlated with tumor aggressiveness and poorer patient outcome, indicating that reduced expression of these genes may participate in malignant progression of RCCs. As is the case when their levels of expression are reduced, these partner genes also may not fully function when involved in chimeric transcripts. These data suggest that generation of chimeric transcripts may participate in renal carcinogenesis by inducing dysfunction of tumor-related genes. (c) 2014 The Authors. Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc.

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Prognostication of patients with clear cell renal cell carcinomas based on quantification of DNA methylation levels of CpG island methylator phenotype marker genes

Tian, Ying, Arai, Eri, Gotoh, Masahiro, Komiyama, Motokiyo, Fujimoto, Hiroyuki, Kanai, Yae

BMC CANCER (BIOMED CENTRAL LTD) 14 ( 1 ) 64 - 72 2014.10

Research paper (scientific journal), ISSN 1471-2407

Background: The CpG island methylator phenotype (CIMP) of clear cell renal cell carcinomas (ccRCCs) is characterized by accumulation of DNA methylation at CpG islands and poorer patient outcome. The aim of this study was to establish criteria for prognostication of patients with ccRCCs using the ccRCC-specific CIMP marker genes.
Methods: DNA methylation levels at 299 CpG sites in the 14 CIMP marker genes were evaluated quantitatively in tissue specimens of 88 CIMP-negative and 14 CIMP-positive ccRCCs in a learning cohort using the MassARRAY system. An additional 100 ccRCCs were also analyzed as a validation cohort.
Results: Receiver operating characteristic curve analysis showed that area under the curve values for the 23 CpG units including the 32 CpG sites in the 7 CIMP-marker genes, i.e. FAM150A, ZNF540, ZNF671, ZNF154, PRAC, TRH and SLC13A5, for discrimination of CIMP-positive from CIMP-negative ccRCCs were larger than 0.95. Criteria combining the 23 CpG units discriminated CIMP-positive from CIMP-negative ccRCCs with 100% sensitivity and specificity in the learning cohort. Cancer-free and overall survival rates of patients with CIMP-positive ccRCCs diagnosed using the criteria combining the 23 CpG units in a validation cohort were significantly lower than those of patients with CIMP-negative ccRCCs (P = 1.41 x 10(-5) and 2.43 x 10(-13), respectively). Patients with CIMP-positive ccRCCs in the validation cohort had a higher likelihood of disease-related death (hazard ratio, 75.8; 95% confidence interval, 7.81 to 735; P = 1.89 x 10(-4)) than those with CIMP-negative ccRCCs.
Conclusions: The established criteria are able to reproducibly diagnose CIMP-positive ccRCCs and may be useful for personalized medicine for patients with ccRCCs.

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Histopathological characteristics of hypervascular cholangiocellular carcinoma as an early stage of cholangiocellular carcinoma

Sato, Yuya, Ojima, Hidenori, Onaya, Hiroaki, Mori, Taisuke, Hiraoka, Nobuyoshi, Kishi, Yoji, Nara, Satoshi, Esaki, Minoru, Shimada, Kazuaki, Kosuge, Tomoo, Sugihara, Kenich, Kanai, Yae

HEPATOLOGY RESEARCH (WILEY-BLACKWELL) 44 ( 11 ) 1119 - 1129 2014.10

Research paper (scientific journal), ISSN 1386-6346

AimPrognosis of hypervascular cholangiocellular carcinoma (h-CCC) is reportedly better than that of ordinary hypovascular CCC (o-CCC). The aim of this study is to clarify the histopathological characteristics of h-CCC.
MethodsOn the basis of the findings in the arterial phase of contrast-enhanced computed tomography, 16 cases of mass-forming-type CCC were divided into two groups (h-CCC, n=8; o-CCC, n=8). Areas of high (Area H-a) and low (Area H-b) attenuation in h-CCC cases and areas of low attenuation in o-CCC cases (Area O) were delineated. These areas were then evaluated histopathologically to determine the proportion of tumor cells, fibrous stroma, arterial vessel density, and immunohistochemical expression of Vascular endothelial growth factor; angiopoietin-2; cytokeratin 7, CK19, SOX9 and SOX17 genes; epithelial cell adhesion molecule; and the Bmi-1, Ki-67, epithelial membrane antigen and polyclonal carcinoembryonic antigen.
ResultsThe areal ratio of tumor cells decreased and that of fibrous stroma increased in the following order: Area H-a, Area H-b and Area O. Values for AVD and neural cell adhesion molecule positivity rate were significantly higher in Area H-a than in Areas H-b or O. Expressions of vascular endothelial growth factor and angiopoietin-2 were significantly higher in Areas H-a and H-b than in Area O. The Ki-67 labeling index increased in the following order: Area H-a, Area H-b and Area O.
ConclusionA high areal ratio of tumor cells and AVD as well as a high expression of stem cells and angiogenic markers were observed in cases of h-CCC, whereas the areal ratio of fibrous stroma and malignant potential were low. These results suggest that h-CCC may represent the early stage of CCC.

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Silencing of microRNA-122 is an early event during hepatocarcinogenesis from non-alcoholic steatohepatitis

Takaki, Yoko, Saito, Yoshimasa, Takasugi, Azusa, Toshimitsu, Kohta, Yamada, Shoji, Muramatsu, Toshihide, Kimura, Masaki, Sugiyama, Kazuo, Suzuki, Hiromu, Arai, Eri, Ojima, Hidenori, Kanai, Yae, Saito, Hidetsugu

CANCER SCIENCE (WILEY-BLACKWELL) 105 ( 10 ) 1254 - 1260 2014.10

Research paper (scientific journal), ISSN 1347-9032

Non-alcoholic steatohepatitis (NASH) has emerged as a common cause of chronic liver disease and virus-independent hepatocellular carcinoma (HCC) in patients with obesity, diabetes, and metabolic syndrome. To reveal the molecular mechanism underlying hepatocarcinogenesis from NASH, microRNA (miRNA) expression profiles were analyzed in STAM mice, a NASH-HCC animal model. MicroRNA expression was also examined in 42 clinical samples of HCC tissue. Histopathological images of the liver of STAM mice at the ages of 6, 8, 12, and 18weeks showed findings compatible with fatty liver, NASH, liver cirrhosis (LC), and HCC, respectively. Expression of miR-122 in non-tumor LC at the age of 18weeks was significantly lower than that in LC at the age of 12weeks. Expression of miR-122 was further decreased in HCCs relative to non-tumor LC at the age of 18weeks. Expression of miR-122 was also decreased in clinical samples of liver tissue showing macrovesicular steatosis and HCC, being consistent with the findings in the NASH model mice. DNA methylation analysis revealed that silencing of miR-122 was not mediated by DNA hypermethylation of the promoter region. These results suggest that silencing of miR-122 is an early event during hepatocarcinogenesis from NASH, and that miR-122 could be a novel molecular marker for evaluating the risk of HCC in patients with NASH.

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Multilayer-omics analysis of renal cell carcinoma, including the whole exome, methylome and transcriptome

Arai, Eri, Sakamoto, Hiromi, Ichikawa, Hitoshi, Totsuka, Hirohiko, Chiku, Suenori, Gotoh, Masahiro, Mori, Taisuke, Nakatani, Tamao, Ohnami, Sumiko, Nakagawa, Tohru, Fujimoto, Hiroyuki, Wang, Linghua, Aburatani, Hiroyuki, Yoshida, Teruhiko, Kanai, Yae

INTERNATIONAL JOURNAL OF CANCER (WILEY-BLACKWELL) 135 ( 6 ) 1330 - 1342 2014.09

Research paper (scientific journal), ISSN 0020-7136

The aim of this study was to identify pathways that have a significant impact during renal carcinogenesis. Sixty-seven paired samples of both noncancerous renal cortex tissue and cancerous tissue from patients with clear cell renal cell carcinomas (RCCs) were subjected to whole-exome, methylome and transcriptome analyses using Agilent SureSelect All Exon capture followed by sequencing on an Illumina HiSeq 2000 platform, Illumina Infinium HumanMethylation27 BeadArray and Agilent Sure-Print Human Gene Expression microarray, respectively. Sanger sequencing and quantitative reverse transcription-PCR were performed for technical verification. MetaCore software was used for pathway analysis. Somatic nonsynonymous single-nucleotide mutations, insertions/deletions and intragenic breaks of 2,153, 359 and 8 genes were detected, respectively. Mutations of GCN1L1, MED12 and CCNC, which are members of CDK8 mediator complex directly regulating beta-catenin-driven transcription, were identified in 16% of the RCCs. Mutations of MACF1, which functions in the Wnt/beta-catenin signaling pathway, were identified in 4% of the RCCs. A combination of methylome and transcriptome analyses further highlighted the significant role of the Wnt/beta-catenin signaling pathway in renal carcinogenesis. Genetic aberrations and reduced expression of ERC2 and ABCA13 were frequent in RCCs, and MTOR mutations were identified as one of the major disrupters of cell signaling during renal carcinogenesis. Our results confirm that multilayer-omics analysis can be a powerful tool for revealing pathways that play a significant role in carcinogenesis.

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Epigenetic clustering of lung adenocarcinomas based on DNA methylation profiles in adjacent lung tissue: Its correlation with smoking history and chronic obstructive pulmonary disease

Sato, Takashi, Arai, Eri, Kohno, Takashi, Takahashi, Yoriko, Miyata, Sayaka, Tsuta, Koji, Watanabe, Shun-ichi, Soejima, Kenzo, Betsuyaku, Tomoko, Kanai, Yae

INTERNATIONAL JOURNAL OF CANCER (WILEY-BLACKWELL) 135 ( 2 ) 319 - 334 2014.07

Research paper (scientific journal), ISSN 0020-7136

The aim of this study was to clarify the significance of DNA methylation alterations during lung carcinogenesis. Infinium assay was performed using 139 paired samples of non-cancerous lung tissue (N) and tumorous tissue (T) from a learning cohort of patients with lung adenocarcinomas (LADCs). Fifty paired N and T samples from a validation cohort were also analyzed. DNA methylation alterations on 1,928 probes occurred in N samples relative to normal lung tissue from patients without primary lung tumors, and were inherited by, or strengthened in, T samples. Unsupervised hierarchical clustering using DNA methylation levels in N samples on all 26,447 probes subclustered patients into Cluster I (n = 32), Cluster II (n = 35) and Cluster III (n = 72). LADCs in Cluster I developed from the inflammatory background in chronic obstructive pulmonary disease (COPD) in heavy smokers and were locally invasive. Most patients in Cluster II were non-smokers and had a favorable outcome. LADCs in Cluster III developed in light smokers were most aggressive (frequently showing lymphatic and blood vessel invasion, lymph node metastasis and an advanced pathological stage), and had a poor outcome. DNA methylation levels of hallmark genes for each cluster, such as IRX2, HOXD8, SPARCL1, RGS5 and EI24, were again correlated with clinicopathological characteristics in the validation cohort. DNA methylation profiles reflecting carcinogenetic factors such as smoking and COPD appear to be established in non-cancerous lung tissue from patients with LADCs and may determine the aggressiveness of tumors developing in individual patients, and thus patient outcome.
What's new? While genetic abnormalities are well studied in human cancers, epigenetic changes, especially in the early stages of carcinogenesis, remain largely unknown. Here, the authors perform a genome-wide analysis focusing on DNA methylation profiles in normal lung tissue adjacent to lung adenocarcinomas. Using single-CpG-resolution Infinium assays, they identify distinct DNA methylation profiles clustering with specific risk factors such as cigarette smoking, inflammation and chronic obstructive pulmonary disease. The authors speculate that these epigenetic profiles detected in the neighboring cells may influence the aggressiveness of tumors developing in individual patients and may thus help predict disease outcome.

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Early venous return in hepatic angiomyolipoma due to an intratumoral structure resembling an arteriovenous fistula

Iwao, Yasuhito, Ojima, Hidenori, Onaya, Hiroaki, Sakamoto, Yoshihiro, Kishi, Yoji, Nara, Satoshi, Esaki, Minoru, Mizuguchi, Yasunori, Ushigome, Masahiko, Asahina, Daisuke, Hiraoka, Nobuyoshi, Shimada, Kazuaki, Kosuge, Tomoo, Kanai, Yae

HEPATOLOGY RESEARCH (WILEY-BLACKWELL) 44 ( 6 ) 700 - 706 2014.06

Research paper (scientific journal), ISSN 1386-6346

Early venous return (EVR) is an important radiological feature of hepatic angiomyolipoma (HAML) that can aid in differential diagnosis, but the pathogenic mechanisms of EVR have yet to be elucidated. We present the first HAML case for which a probable mechanism for EVR is described. The patient was a 46-year-old woman, who had a growing 6-cm tumor with EVR in segment 3 of the liver as revealed by dynamic contrast-enhanced computed tomography. Left hepatic lobectomy was performed to prevent tumor rupture. Histopathological and immunohistochemical analyses of the excised tumor indicated HAML. Successive microsections of the tumor were stained with hematoxylin-eosin and Victoria blue to visualize the vascular structure within and around the tumor. These analyses led to three major findings. First, many well-defined thick-walled vessels, such as arteries, were found entering the tumor. Second, many thick-walled vessels within the tumor were connected directly to thin-walled vessels, resembling arteriovenous fistulae. Finally, thin-walled intratumoral vessels were connected directly to the hepatic vein. These histological findings suggested that the rich arterial flow into the tumor was being rapidly drained into the hepatic vein through intratumoral arteriovenous connections. We also detected these same anomalous circulatory pathways in tissue sections from three of four additional HAML cases with EVR. Aberrant arteriovenous fistulae within the tumor may account for many cases of EVR in HAML patients.

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PHLDA3 is a novel tumor suppressor of pancreatic neuroendocrine tumors

Ohki, Rieko, Saito, Kozue, Chen, Yu, Kawase, Tatsuya, Hiraoka, Nobuyoshi, Saigawa, Raira, Minegishi, Maiko, Aita, Yukie, Yanai, Goichi, Shimizu, Hiroko, Yachida, Shinichi, Sakata, Naoaki, Doi, Ryuichiro, Kosuge, Tomoo, Shimada, Kazuaki, Tycko, Benjamin, Tsukada, Toshihiko, Kanai, Yae, Sumi, Shoichiro, Namiki, Hideo, Taya, Yoichi, Shibata, Tatsuhiro, Nakagama, Hitoshi

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA (NATL ACAD SCIENCES) 111 ( 23 ) E2404 - E2413 2014.06

Research paper (scientific journal), ISSN 0027-8424

The molecular mechanisms underlying the development of pancreatic neuroendocrine tumors (PanNETs) have not been well defined. We report here that the genomic region of the PHLDA3 gene undergoes loss of heterozygosity (LOH) at a remarkably high frequency in human PanNETs, and this genetic change is correlated with disease progression and poor prognosis. We also show that the PHLDA3 locus undergoes methylation in addition to LOH, suggesting that a two-hit inactivation of the PHLDA3 gene is required for PanNET development. We demonstrate that PHLDA3 represses Akt activity and Akt-regulated biological processes in pancreatic endocrine tissues, and that PHLDA3-deficient mice develop islet hyperplasia. In addition, we show that the tumor-suppressing pathway mediated by MEN1, a well-known tumor suppressor of PanNETs, is dependent on the pathway mediated by PHLDA3, and inactivation of PHLDA3 and MEN1 cooperatively contribute to PanNET development. Collectively, these results indicate the existence of a novel PHLDA3-mediated pathway of tumor suppression that is important in the development of PanNETs.

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Frequent Lack of GNAS Mutations in Colorectal Adenocarcinoma Associated with GNAS-Mutated Villous Adenoma

Sekine, Shigeki, Ogawa, Reiko, Oshiro, Taihei, Kanemitsu, Yukihide, Taniguchi, Hirokazu, Kushima, Ryoji, Kanai, Yae

GENES CHROMOSOMES & CANCER (WILEY-BLACKWELL) 53 ( 4 ) 366 - 372 2014.04

Research paper (scientific journal), ISSN 1045-2257

Colorectal villous adenoma is thought to be associated with a high risk of progression to adenocarcinoma. To better characterize the genetic alterations involved in colorectal carcinogenesis related to villous adenoma, we analyzed mutations in APC, BRAF, KRAS, TP53, and GNAS in 12 colorectal adenocarcinomas associated with villous adenomas. APC, KRAS, and BRAF mutations were identified in five, 11, and one lesion, respectively, and most of these mutations were shared between the villous adenoma and the adenocarcinoma components in the respective lesions, except in one lesion with APC mutations and in two lesions with KRAS mutations. TP53 mutations were observed exclusively in four adenocarcinoma components, consistent with their role in the progression from adenoma to adenocarcinoma. Activating GNAS mutations were found in nine villous adenomas; however, unexpectedly, these mutations were shared only in three associated adenocarcinomas. Notably, all six adenocarcinomas with discordant GNAS mutation statuses were nonmucinous type, whereas all the other adenocarcinomas, including three adenocarcinomas associated with GNAS wild-type villous adenomas, were mucinous type. The current study suggests that GNAS-mutated villous adenomas may not necessarily be direct precursors of associated adenocarcinomas. At the same time, our observations support the role of activating GNAS mutations in increased mucin production in colorectal neoplasms. © 2014 Wiley Periodicals, Inc.

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Lobular Endocervical Glandular Hyperplasia Is a Neoplastic Entity With Frequent Activating GNAS Mutations

Matsubara, Akiko, Sekine, Shigeki, Ogawa, Reiko, Yoshida, Masayuki, Kasamatsu, Takahiro, Tsuda, Hitoshi, Kanai, Yae

AMERICAN JOURNAL OF SURGICAL PATHOLOGY (LIPPINCOTT WILLIAMS & WILKINS) 38 ( 3 ) 370 - 376 2014.03

Research paper (scientific journal), ISSN 0147-5185

To clarify the significance of GNAS mutations in cervical tumorigenesis, we performed mutational analyses in a total of 154 lesions and in 22 normal tissues of the uterine cervix. Activating GNAS mutations were found in 8 of the 19 lobular endocervical glandular hyperplasias (LEGH; 42%) and 4 of the 79 endocervical-type mucinous adenocarcinomas (5%) but were never seen in the normal endocervical tissue, minimal deviation adenocarcinomas, endometrioid adenocarcinomas, or squamous cell carcinomas. We further examined the presence of human papillomavirus (HPV) DNA and p16 expression to probe the relationship between GNAS mutations and HPV infection in LEGHs and carcinomas. All the GNAS-mutated LEGHs were negative for HPV DNA and p16 expression, whereas all the GNAS-mutated adenocarcinomas were positive for HPV DNA and/or p16 expression, implicating GNAS mutations in the development of LEGH and a minor subset of HPV-related cervical adenocarcinomas. Additional mutational analyses of LEGH identified KRAS and STK11 mutations in 1 and 2 cases, respectively. The GNAS, KRAS, and STK11 mutations were mutually exclusive; thus, a total of 11 LEGHs (58%) had 1 of these genetic alterations. Although LEGH has been regarded as a metaplastic lesion, the frequent presence of genetic alterations suggests a neoplastic nature.

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Association of Pancreatic Fatty Infiltration With Pancreatic Ductal Adenocarcinoma

Hori, Mika, Takahashi, Mami, Hiraoka, Nobuyoshi, Yamaji, Taiki, Mutoh, Michihiro, Ishigamori, Rikako, Furuta, Koh, Okusaka, Takuji, Shimada, Kazuaki, Kosuge, Tomoo, Kanai, Yae, Nakagama, Hitoshi

CLINICAL AND TRANSLATIONAL GASTROENTEROLOGY (NATURE PUBLISHING GROUP) 5 2304 - 2315 2014.03

Research paper (scientific journal), ISSN 2155-384X

OBJECTIVES: Fatty infiltration (FI) in the pancreas is positively correlated with high body mass index (BMI) or obesity, and the prevalence of diabetes mellitus (DM), which are well-known risk factors of pancreatic cancer. However, the association of FI in the pancreas with pancreatic cancer is unclear. Recently, we have shown that Syrian golden hamsters feature FI of the pancreas, the severity of which increases along with the progression of carcinogenesis induced by a chemical carcinogen. To translate the results to a clinical setting, we investigated whether FI in the pancreas is associated with pancreatic cancer in a series of patients who had undergone pancreatoduodenectomy.
METHODS: In the series, we identified 102 cases with pancreatic ductal adenocarcinoma (PDAC) and 85 controls with cancers except for PDAC. The degree of FI was evaluated histopathologically from the area occupied by adipocytes in pancreas sections, and was compared between the cases and controls.
RESULTS: The degree of FI in the pancreas was significantly higher in cases than in controls (median 26 vs. 15%, P<0.001) and positively associated with PDAC, even after adjustment for BMI, prevalence of DM and other confounding factors (odds ratio (OR), 6.1; P<0.001). BMI was identified as the most significantly associated factor with FI in the pancreas.
CONCLUSIONS: There is a positive correlation between FI in the pancreas and pancreatic cancer.

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Multilayer-omics analyses of human cancers: exploration of bionnarkers and drug targets based on the activities of the International Human Epigenome Consortium

Kanai, Yae, Arai, Eri

FRONTIERS IN GENETICS 5 1330 - 1342 2014.02

Research paper (scientific journal), ISSN 1664-8021

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Loss of NDRG2 expression activates PI3K-AKT signalling via PTEN phosphorylation in ATLL and other cancers

Nakahata, Shingo, Ichikawa, Tomonaga, Maneesaay, Phudit, Saito, Yusuke, Nagai, Kentaro, Tamura, Tomohiro, Manachai, Nawin, Yamakawa, Norio, Hamasaki, Makoto, Kitabayashi, Issay, Arai, Yasuhito, Kanai, Yae, Taki, Tomohiko, Abe, Takaya, Kiyonari, Hiroshi, Shimoda, Kazuya, Ohshima, Koichi, Horii, Akira, Shima, Hiroshi, Taniwaki, Masafumi, Yamaguchi, Ryoji, Morishita, Kazuhiro

NATURE COMMUNICATIONS (NATURE PUBLISHING GROUP) 5 91 - 93 2014.02

Research paper (scientific journal), ISSN 2041-1723

Constitutive phosphatidylinositol 3-kinase (PI3K)-AKT activation has a causal role in adult T-cell leukaemia-lymphoma (ATLL) and other cancers. ATLL cells do not harbour genetic alterations in PTEN and PI3KCA but express high levels of PTEN that is highly phosphorylated at its C-terminal tail. Here we report a mechanism for the N-myc downstream-regulated gene 2 (NDRG2)-dependent regulation of PTEN phosphatase activity via the dephosphorylation of PTEN at the Ser380, Thr382 and Thr383 cluster within the C-terminal tail. We show that NDRG2 is a PTEN-binding protein that recruits protein phosphatase 2A (PP2A) to PTEN. The expression of NDRG2 is frequently downregulated in ATLL, resulting in enhanced phosphorylation of PTEN at the Ser380/Thr382/Thr383 cluster and enhanced activation of the PI3K-AKT pathway. Given the high incidence of T-cell lymphoma and other cancers in NDRG2-deficient mice, PI3K-AKT activation via enhanced PTEN phosphorylation may be critical for the development of cancer.

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Intraductal dissemination of papillary adenocarcinoma of the ampulla of Vater in the pancreatic duct

Matsubara, Akiko, Nara, Satoshi, Sekine, Shigeki, Ojima, Hidenori, Kosuge, Tomoo, Shimada, Kazuaki, Kushima, Ryoji, Kanai, Yae, Hiraoka, Nobuyoshi

PATHOLOGY INTERNATIONAL (WILEY-BLACKWELL) 64 ( 1 ) 39 - 44 2014.01

Research paper (scientific journal), ISSN 1320-5463

It has been speculated that intraductal dissemination, via the pancreatic duct, bile duct, or mammary duct, is a unique form of cancer cell spread. However, clinical evidence to confirm this form of dissemination has been lacking. Here we report a case of papillary adenocarcinoma of the ampulla of Vater in which retrograde dissemination to the pancreatic duct was strongly suggested. A 79-year-old woman underwent pancreatoduodenectomy for a 22 mm microinvasive papillary adenocarcinoma of the ampulla. Multiple carcinomas in situ were found in the pancreatic duct distant from the ampulla. Seven months later, she underwent a second operation for a recurrent papillary adenocarcinoma at the pancreato-jejunal anastomosis showing exophytic and expansive growth into the jejunal lumen that connected to an intraductal adenocarcinoma in the pancreatic body. None of these tumors showed invasive growth, or vascular or neural invasion, being separate from each other but sharing identical histological, immunohistochemical, and molecular features; papillary growth, a pancreatobiliary phenotype, the same pattern of genomic loss of heterozygosity, and no mutation of the KRAS, TP53, and GNAS genes. These results imply that this papillary adenocarcinoma of the ampulla of Vater had disseminated to the pancreatic duct in a retrograde manner and recurred in the remnant pancreas.

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Clinicopathological characterization of so-called "cholangiocarcinoma with intraductal papillary growth" with respect to "intraductal papillary neoplasm of bile duct (IPNB)"

Kanai Yae

INTERNATIONAL JOURNAL OF CLINICAL AND EXPERIMENTAL PATHOLOGY (E-CENTURY PUBLISHING CORP) 7 ( 6 ) 3112 - 3122 2014

Research paper (scientific journal), ISSN 1936-2625

Cholangiocarcinoma (CC) of the biliary tract occasionally presents a predominant intraductal papillary growth in the bile ducts, called as biliary tract carcinoma (BTC) of papillary growth (PG) and intrahepatic CC (ICC) of intraductal growth (IG) type. Recently, intraductal papillary neoplasm of bile duct (IPNB) has been proposed as a pre- invasive biliary neoplasm. This study was performed to characterize pathologically BTC of PG type and ICC of IG type with respect to IPNB. It was found that 126 of such 154 CCs (81.8%) fulfilled the criteria of IPNB, while the remaining 28 cases showed different histologies, such as tubular adenocarcinoma and carcinosarcoma. These IPNBs occurred in old aged patients with a male predominance, and the left lobe was rather frequently affected in the liver. A majority of these cases were high grade IPNB (43 cases) and invasive IPNB (77 cases), while low grade IPNB was rare (6 cases). Pancreatobiliary type was predominant (48 cases) followed by gastric (30 cases), intestinal (29 cases) and oncocytic (19 cases) types. Mucus hypersecretion was found in 45 cases, and this was frequent in IPNB at the intrahepatic large bile duct and hilar bile ducts but rare at the extrahepatic bile ducts. Interestingly, 36 cases of high grade and invasive IPNBs contained foci of moderately differentiated adenocacinoma within the intraductal papillary tumor. In conclusion, a majority of ICC of IG type and BTC of PG type could be regarded as a IPNB lineage, and clinically detectable IPNBs were already a malignant papillary lesion.

Potential biomarker for human uterine leiomyosarcoma.

Hayashi T, Horiuchi A, Aburatani H, Ishiko O, Yaegashi N, Kanai Y, Zharhary D, Shiozawa T, Tonegawa S, Konishi I.

J Clin Med Res 6 392 - 394 2014

Accepted

Pathological diagnosis of flat epithelial lesions of the biliary tract with emphasis on biliary intraepithelial neoplasia.

Sato Y, Sasaki M, Harada K, Aishima S, Fukusato T, Ojima H, Kanai Y, Kage M, Nakanuma Y, Tsubouchi H; Hepatolithiasis Subdivision of Intractable Hepatobiliary Diseases Study Group of Japan (Chairman, Hirohito Tsubouchi).

J Gastroenterol (SPRINGER JAPAN KK) 49 ( 1 ) 64 - 72 2014

Accepted, ISSN 09441174

Flat epithelial lesions of the biliary tract cannot be detected by the image analysis, and the diagnosis entirely depends on pathological examination. The biliary tract is often affected by inflammatory conditions, and the resultant changes of the biliary epithelium make it difficult to differentiate them from neoplasia. Thus, the pathological diagnosis of biliary flat epithelial lesions can be challenging. In the biliary tract, there are several forms of intraepithelial neoplasia of the flat type, and biliary intraepithelial neoplasia (BilIN) is known as one of such lesions that represent the multistep cholangiocarcinogenesis. In this article, the diagnostic criteria and the differential diagnosis of biliary flat epithelial lesions, particularly focusing on BilIN, were presented and discussed to provide help to advance clinical and research applications of the BilIN system.

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Stem cell self-renewal factors Bmi1 and HMGA2 in head and neck squamous cell carcinoma: clues for diagnosis

Yamazaki, Hiroshi, Mori, Taisuke, Yazawa, Masaki, Maeshima, Akiko M., Matsumoto, Fumihiko, Yoshi-moto, Seiichi, Ota, Yoshihide, Kaneko, Akihiro, Tsuda, Hitoshi, Kanai, Yae

LABORATORY INVESTIGATION (NATURE PUBLISHING GROUP) 93 ( 12 ) 1331 - 1338 2013.12

Research paper (scientific journal), ISSN 0023-6837

Head and neck squamous cell carcinoma (HNSCC) includes both morphological and functional cellular heterogeneity, as would be expected if it arose from dysregulated stem or progenitor cells as opposed to the simple clonal expansion of a mutated cell; however, stemness molecule expression levels and distribution in HNSCC remain unclear. To clarify this, stemness molecule expressions were determined in HNSCC, as well as their properties and prognosis. Two protooncogenic chromatin regulators, Bmi-1 and high-mobility-group A2 (Hmga2), were identified in 12 pair cases of HNSCC tumor regions by comparison with their non-cancerous background tissues using cDNA microarray. Both Bmi-1 and Hmga2 are known to promote stem cell self-renewal by negatively regulating the expressions of Ink4a and Arf tumor suppressors. Despite similar targets, Bmi-1 protein was expressed in an early cancerous region and HMGA2 protein was expressed in a region showing more progression. Similarly, Bmi1 expression had no significance with regard to overall survival (P = 0.67), whereas HMGA2 expression was associated with decreased overall survival (P = 0.05). Quantitative real-time reverse transcription polymerase chain reaction analyses also correlated with protein levels. These findings suggest that Bmi-1 is an early detection marker to distinguish cancerous from non-cancerous regions, whereas HMGA2 is presumed to be a tumor prognosis marker. Among our HNSCC analyses, these stemness molecules expressed fewer primitive rare cells in the tumor than all other cells in the tumor. HNSCC cells with high expression of stemness molecules partly behave like stem cells.

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Bile Acids Increase Levels of MicroRNAs 221 and 222, Leading to Degradation of CDX2 During Esophageal Carcinogenesis

Matsuzaki, Juntaro, Suzuki, Hidekazu, Tsugawa, Hitoshi, Watanabe, Mitsuhiro, Hossain, Sharif, Arai, Eri, Saito, Yoshimasa, Sekine, Shigeki, Akaike, Toshihiro, Kanai, Yae, Mukaisho, Ken-Ichi, Auwerx, Johan, Hibi, Toshifumi

GASTROENTEROLOGY (Gastroenterology) 145 ( 6 ) 1300 - 1311 2013.12

Research paper (scientific journal), ISSN 0016-5085

Background &amp
Aims Bile reflux contributes to development of Barrett's esophagus (BE) and could be involved in its progression to esophageal adenocarcinoma (EAC). We investigated whether bile acids affect levels or functions of microRNAs (MIRs) 221 and 222, which bind to the 3′-UTR of p27Kip1 messenger RNA to inhibit its translation. Reduced p27Kip1 increases degradation of the transcription factor CDX2
levels of CDX2 have been reported to decrease during progression of BE to EAC. Methods We used quantitative reverse transcriptase polymerase chain reaction to compare levels of MIRs 221 and 222 and immunohistochemistry to compare levels of p27Kip1 and CDX2 proteins in areas of BE and EAC from each of 11 patients. We examined the effects of bile acid exposure on levels of MIRs 221 and 222 and CDX2 in EAC cells. We investigated the effects of inhibitors of MIRs 221 and 222 on growth of human EAC xenograft tumors in NOD/SCID/IL-2Rγnull mice. Results Levels of MIRs 221 and 222 increased and levels of p27Kip1 and CDX2 decreased in areas of EAC vs BE. Levels of MIRs 221 and 222 increased, along with activity of nuclear bile acid receptor/farnesoid X receptor (FXR), when cultured cells were exposed to bile acids. Incubation of cells with bile acids increased degradation of CDX2
this process was reduced when cells were also incubated with proteasome inhibitors. Overexpression of MIRs 221 and 222 reduced levels of p27Kip1 and CDX2, and knockdown of these MIRs increased levels of these proteins in cultured cells. Inhibitors of MIRs 221 and 222 increased levels of p27Kip1 and CDX2 in EAC cells and reduced growth of xenograft tumors in NOD/SCID/IL-2Rγnull mice. Conclusions We observed increased levels of MIRs 221 and 222 in human EAC tissues, compared with areas of BE from the same patient. We found that exposure of esophageal cells to bile acids activates FXR and increases levels of MIRs 221 and 222, reducing levels of p27Kip1 and promoting degradation of CDX2 by the proteasome. Our work opened the perspective of therapeutically targeting this pathway either via FXR antagonists or inhibitors of MIRs as a treatment option for BE and EAC. © 2013 by the AGA Institute.

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Ability of preoperative 3.0-Tesla magnetic resonance imaging to predict the absence of side-specific extracapsular extension of prostate cancer

Hara, Tomohiko, Nakanishi, Hiroyuki, Nakagawa, Tohru, Komiyama, Motokiyo, Kawahara, Takashi, Manabe, Tomoko, Miyake, Mototaka, Arai, Eri, Kanai, Yae, Fujimoto, Hiroyuki

INTERNATIONAL JOURNAL OF UROLOGY (WILEY-BLACKWELL) 20 ( 10 ) 993 - 999 2013.10

Research paper (scientific journal), ISSN 0919-8172

ObjectiveRecent studies have shown an improvement in prostate cancer diagnosis with the use of 3.0-Tesla magnetic resonance imaging. We retrospectively assessed the ability of this imaging technique to predict side-specific extracapsular extension of prostate cancer.
MethodsFrom October 2007 to August 2011, prostatectomy was carried out in 396 patients after preoperative 3.0-Tesla magnetic resonance imaging. Among these, 132 (primary sample) and 134 patients (validation sample) underwent 12-core prostate biopsy at the National Cancer Center Hospital of Tokyo, Japan, and at other institutions, respectively. In the primary dataset, univariate and multivariate analyses were carried out to predict side-specific extracapsular extension using variables determined preoperatively, including 3.0-Tesla magnetic resonance imaging findings (T2-weighted and diffusion-weighted imaging). A prediction model was then constructed and applied to the validation study sample.
ResultsMultivariate analysis identified four significant independent predictors (P<0.05), including a biopsy Gleason score of 8, positive 3.0-Tesla diffusion-weighted magnetic resonance imaging findings, 2 positive biopsy cores on each side and a maximum percentage of positive cores 31% on each side. The negative predictive value was 93.9% in the combination model with these four predictors, meanwhile the positive predictive value was 33.8%. Good reproducibility of these four significant predictors and the combination model was observed in the validation study sample.
ConclusionsThe side-specific extracapsular extension prediction by the biopsy Gleason score and factors associated with tumor location, including a positive 3.0-Tesla diffusion-weighted magnetic resonance imaging finding, have a high negative predictive value, but a low positive predictive value.

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Bile duct carcinoma involving the common channel associated with pancreaticobiliary maljunction shows an extension pattern similar to ductal carcinoma of the pancreas

Yoshida, Nao, Esaki, Minoru, Kishi, Yoji, Shimada, Kazuaki, Ojima, Hidenori, Kanai, Yae, Hiraoka, Nobuyoshi

PATHOLOGY INTERNATIONAL (WILEY-BLACKWELL) 63 ( 8 ) 415 - 418 2013.08

Research paper (scientific journal), ISSN 1320-5463

Biliary tract cancer occurs frequently in patients with pancreaticobiliary maljunction (PBM), although no details of its clinicopathological characteristics have been reported. Here we describe a case of bile duct (BD) cancer that developed in association with PBM. This BD cancer involved the common channel, extended to the main pancreatic duct (MPD) via the common channel, and invaded the pancreatic parenchyma, where its growth and spread, and features of its recurrence, were similar to those of ductal carcinoma of the pancreas. We assumed that MPD extension of BD cancer via the common channel was the reason for its deep spread to the pancreas, since BD cancer usually spreads along the BD and rarely reaches the common channel of the ampulla of Vater. During the follow-up of patients with PBM, attention should be paid to involvement of the common channel by BD cancer and also to cancer developing silently in the pancreas after extrahepatic BD resection.

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SOX10 is a novel marker of acinus and intercalated duct differentiation in salivary gland tumors: a clue to the histogenesis for tumor diagnosis

Ohtomo, Rie, Mori, Taisuke, Shibata, Shinsuke, Tsuta, Koji, Maeshima, Akiko M., Akazawa, Chihiro, Watabe, Yukio, Honda, Kazufumi, Yamada, Tesshi, Yoshimoto, Seiichi, Asai, Masao, Okano, Hideyuki, Kanai, Yae, Tsuda, Hitoshi

MODERN PATHOLOGY (Modern Pathology) 26 ( 8 ) 1041 - 1050 2013.08

Research paper (scientific journal), ISSN 0893-3952

Salivary gland tumors are relatively rare and morphologically diverse and heterogeneous tumors
therefore, histogenesis-based tumor markers are sorely needed to aid in diagnosing and determining the cell type of origin. SRY-related HMG-box 10 (SOX10) protein is a transcription factor known to be crucial in the specification of the neural crest and maintenance of Schwann cells and melanocytes. In addition, positive expression has also been implicated in the major salivary gland. Here, we examined SOX10 expression in various salivary gland tumors to correlate this expression with myoepithelial markers. Overall, 76 malignant and 14 benign tumors were examined. SOX10 expression clearly delineated two distinct subtypes of human salivary gland tumors
acinic cell carcinomas, adenoid cystic carcinomas, epithelial-myoepithelial carcinomas, myoepithelial carcinomas, and pleomorphic adenomas, including the pleomorphic adenoma component of carcinoma, were SOX10 positive, while salivary duct carcinomas, mucoepidermoid carcinomas, an oncocytic carcinoma, Oncocytomas, and Warthin tumors were SOX10 negative. Also, SOX10 was expressed in solid-type or non-specific morphology salivary gland tumors, but was not expressed in poorly differentiated squamous cell carcinomas. In normal human salivary gland tissue, SOX10 expression was specific to the nuclei of acini and both luminal and abluminal cells of intercalated ducts but not in other sites. Moreover, the murine model suggested that SOX10 continued to be expressed from the developmental stage to adulthood in the acinar and both luminal and abluminal intercalated ducts in the major salivary gland. Thus, SOX10 is a novel marker for diagnosing and understanding the histogenesis of salivary gland tumors. © 2013 USCAP, Inc. All rights reserved.

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Tumor immunoediting, from T cell-mediated immune surveillance to tumor-escape of uterine leiomyosarcoma

Takuma Hayashi, Akiko Horiuchi, Kenji Sano, Nobuyoshi Hiraoka, Tomoyuki Ichimura, Osamu Ishiko, Yae Kanai, Nobuo Yaegashi, Hiroyuki Aburatani, Tanri Shiozawa, Susumu Tonegawa, Ikuo Konishi

Journal of Vaccines and Vaccination (Journal of Vaccines and Vaccination) 4 ( SPL.ISSUE ) 2013.07

ISSN 2157-7560

The majority of smooth muscle tumors found in the uterus are benign, but uterine leiomyosarcomas (LMSs) are extremely malignant, with high rates of recurrence and metastasis. The development of gynecologic tumors is often correlated with female hormone secretion
however, the development of uterine LMS is not substantially correlated with hormonal conditions, and the risk factors are not clearly understood. The presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules is important for tumor rejection by cytotoxic T-lymphocytes (CTLs). Such antigenic peptides are generated as a result of the degradation of intracellular proteins by the proteasome pathway, a process that is influenced by the interferon (IFN)-γ-inducible low molecular mass polypeptide-2 (LMP2) subunit of the 20S proteasome. Homozygous deficient mice for LMP2 are now known to spontaneously develop uterine LMS. LMP2 expression is reportedly absent in human uterine LMS, but present in human myometrium. Further studies revealed a few infiltrating CD56+ NK cells in human uterine LMS tissues. This review aims at summarizing recent insights into the regulation of NK cell function and the T cell-mediated immune system as tumor immune surveillance, first attempts to exploit NK cell activation to improve immunity to tumors. © 2012 Hayashi T, et al.

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Pancreatic Intraglandular Metastasis Predicts Poorer Outcome in Postoperative Patients With Pancreatic Ductal Carcinoma

Oguro, Seiji, Shimada, Kazuaki, Ino, Yoshinori, Esaki, Minoru, Nara, Satoshi, Kishi, Yoji, Kosuge, Tomoo, Kanai, Yae, Hiraoka, Nobuyoshi

AMERICAN JOURNAL OF SURGICAL PATHOLOGY (LIPPINCOTT WILLIAMS & WILKINS) 37 ( 7 ) 1030 - 1038 2013.07

Research paper (scientific journal), ISSN 0147-5185

Intraorgan metastasis of a primary cancer within the organ of origin, such as intrahepatic metastasis of hepatocellular carcinoma, is one of the key features for clinicopathologic staging of the cancer. Pancreatic intraglandular metastasis (P-IM) of pancreatic ductal carcinoma (PDC) is encountered occasionally but has not yet been evaluated. The aim of this study was to investigate the clinicopathologic characteristics and prognostic value of P-IM in patients with PDC. The histopathologic features of 393 consecutive patients with PDC who had undergone pancreatic resection at the National Cancer Center Hospital, Tokyo, between 2003 and 2010 were reviewed. For the purposes of the study, P-IM was defined as an independent tumor showing histopathologic features similar to those of the primary one. Twenty-six cases of P-IM were identified in 21 (5.3%) of the reviewed patients. The incidence of P-IM at each stage of the TNM classification was 0% (0/7) at stage IA, 17% (1/6) at stage IB, 5% (5/92) at stage IIA, 4% (11/252) at stage IIB, 0% (0/1) at stage III, and 11% (4/35) at stage IV. Univariate survival analysis showed that both overall survival and disease-free survival for patients with P-IM were significantly shorter than for those without P-IM (P < 0.001 and P = 0.019, respectively). Multivariate survival analysis showed that P-IM was significantly correlated with shorter overall survival (P = 0.002; hazard ratio = 2.239; 95% confidence interval: 1.328-3.773). Our findings suggest that the presence of P-IM in patients with PDC is an independent prognosticator and may represent aggressive tumor behavior.

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Diagnostic markers of urothelial cancer based on DNA methylation analysis

Chihara, Yoshitomo, Kanai, Yae, Fujimoto, Hiroyuki, Sugano, Kokichi, Kawashima, Kiyotaka, Liang, Gangning, Jones, Peter A., Fujimoto, Kiyohide, Kuniyasu, Hiroki, Hirao, Yoshihiko

BMC CANCER (BIOMED CENTRAL LTD) 13 794 - 798 2013.06

Research paper (scientific journal), ISSN 1471-2407

Background: Early detection and risk assessment are crucial for treating urothelial cancer (UC), which is characterized by a high recurrence rate, and necessitates frequent and invasive monitoring. We aimed to establish diagnostic markers for UC based on DNA methylation.
Methods: In this multi-center study, three independent sample sets were prepared. First, DNA methylation levels at CpG loci were measured in the training sets (tumor samples from 91 UC patients, corresponding normal-appearing tissue from these patients, and 12 normal tissues from age-matched bladder cancer-free patients) using the Illumina Golden Gate methylation assay to identify differentially methylated loci. Next, these methylated loci were validated by quantitative DNA methylation by pyrosequencing, using another cohort of tissue samples (Tissue validation set). Lastly, methylation of these markers was analyzed in the independent urine samples (Urine validation set). ROC analysis was performed to evaluate the diagnostic accuracy of these 12 selected markers.
Results: Of the 1303 CpG sites, 158 were hyper ethylated and 356 were hypo ethylated in tumor tissues compared to normal tissues. In the panel analysis, 12 loci showed remarkable alterations between tumor and normal samples, with 94.3% sensitivity and 97.8% specificity. Similarly, corresponding normal tissue could be distinguished from normal tissues with 76.0% sensitivity and 100% specificity. Furthermore, the diagnostic accuracy for UC of these markers determined in urine samples was high, with 100% sensitivity and 100% specificity.
Conclusion: Based on these preliminary findings, diagnostic markers based on differential DNA methylation at specific loci can be useful for non-invasive and reliable detection of UC and epigenetic field defect.

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Prognostic Risk Stratification of Patients with Urothelial Carcinoma of the Bladder with Recurrence After Radical Cystectomy

Nakagawa, Tohru, Hara, Tomohiko, Kawahara, Takashi, Ogata, Yoshihiko, Nakanishi, Hiroyuki, Komiyama, Motokiyo, Arai, Eri, Kanai, Yae, Fujimoto, Hiroyuki

JOURNAL OF UROLOGY (Journal of Urology) 189 ( 4 ) 1275 - 1281 2013.04

Research paper (scientific journal), ISSN 0022-5347

Purpose: We identify clinicopathological variables predicting overall survival in patients with recurrent bladder urothelial carcinoma after radical cystectomy. Materials and Methods: We retrospectively collected data on 114 patients treated with radical cystectomy for bladder urothelial carcinoma who subsequently had remote metastasis and/or local recurrence. The Kaplan-Meier method with the log rank test and multivariate Cox regression models were used to address overall survival after recurrence. Results: During followup 99 of the 114 patients died. Median survival in the 114 patients was 11.2 months. One and 3-year overall survival rates were 48.0% and 12.1%, respectively. On multivariate analysis independent predictors of poorer overall survival included less than 1 year to recurrence, symptoms at recurrence, 2 or more metastatic organs at recurrence, high serum C-reactive protein, high lactate dehydrogenase, no post-recurrence platinum based chemotherapy and no metastasectomy. Based on the 4 variables (time to recurrence, symptoms, number of metastatic organs and C-reactive protein), we constructed a risk model predicting post-recurrence overall survival that classified patients into 3 groups with significantly different overall survival (p &lt
0.0001). Conclusions: Our data confirm that recurrent urothelial carcinoma after radical cystectomy is a highly aggressive, lethal disease. Seven clinicopathological factors were identified that predicted post-recurrence overall survival. Our risk model based on the 4 variables could be useful to provide relevant prognostic information to patients and physicians, and better stratify patients in clinical trials. © 2013 American Urological Association Education and Research, Inc.

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The tumor suppressor microRNA-29c is downregulated and restored by celecoxib in human gastric cancer cells

Saito, Yoshimasa, Suzuki, Hidekazu, Imaeda, Hiroyuki, Matsuzaki, Juntaro, Hirata, Kenro, Tsugawa, Hitoshi, Hibino, Sana, Kanai, Yae, Saito, Hidetsugu, Hibi, Toshifumi

INTERNATIONAL JOURNAL OF CANCER (International Journal of Cancer) 132 ( 8 ) 1751 - 1760 2013.04

Research paper (scientific journal), ISSN 0020-7136

MicroRNAs (miRNAs) are small noncoding RNAs that function as endogenous silencers of target genes and play critical roles during carcinogenesis. The selective cyclooxygenase-2 (COX-2) inhibitor celecoxib has been highlighted as a potential drug for treatment of gastrointestinal tumors. The aim of this study was to investigate the role of miRNAs in gastric carcinogenesis and the feasibility of a new therapeutic approach for gastric cancer. miRNA expression profiles were examined in 53 gastric tumors including gastric adenomas (atypical epithelia), early gastric cancers and advanced gastric cancers and in gastric cancer cells treated with celecoxib. miRNA microarray analysis revealed that miR-29c was significantly downregulated in gastric cancer tissues relative to nontumor gastric mucosae. miR-29c was significantly activated by celecoxib in gastric cancer cells. Downregulation of miR-29c was associated with progression of gastric cancer and was more prominent in advanced gastric cancers than in gastric adenomas and early gastric cancer. In addition, expression of the oncogene Mcl-1, a target of miR-29c, was significantly increased in gastric cancer tissues relative to nontumor gastric mucosae. Activation of miR-29c by celecoxib induced suppression of Mcl-1 and apoptosis in gastric cancer cells. These results suggest that downregulation of the tumor suppressor miR-29c plays critical roles in the progression of gastric cancer. Selective COX-2 inhibitors may have clinical promise for the treatment of gastric cancer via restoration of miR-29c. What's new? MicroRNAs (miRNAs) play critical roles in carcinogenesis and may be valuable therapeutic targets for malignant disease. Here, the miRNA tumor suppressor miR-29c was found to be significantly downregulated in gastric cancer cells and to be reactivated by the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib. Reactivation led to miR-29c-induced suppression of the anti-apoptotic protein Mcl-1. The data reveal that selective COX-2 inhibitors may have clinical promise for the treatment of gastric cancer via restoration of miR-29c. Copyright © 2012 UICC.

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Clinical impact of c-Met expression and its gene amplification in hepatocellular carcinoma

Kondo, Shunsuke, Ojima, Hidenori, Tsuda, Hitoshi, Hashimoto, Jun, Morizane, Chigusa, Ikeda, Masafumi, Ueno, Hideki, Tamura, Kenji, Shimada, Kazuaki, Kanai, Yae, Okusaka, Takuji

INTERNATIONAL JOURNAL OF CLINICAL ONCOLOGY (SPRINGER JAPAN KK) 18 ( 2 ) 207 - 213 2013.04

Research paper (scientific journal), ISSN 1341-9625

c-Met is an oncogene encoding a receptor for hepatocyte growth factor and, as such, plays a key role in hepatocellular carcinomas (HCC). We evaluated c-Met protein expression and its gene amplification in order to assess whether they were related to tumor recurrence and survival rates among patients who had undergone tumor resection.
We used the polymer-based method to perform an immunohistochemistry analysis of c-Met expression on 59 formalin-fixed, paraffin-embedded sections of surgical specimens. c-Met gene amplification was investigated with fluorescence in-situ hybridization. Kaplan-Meier methods and Cox proportional hazards models were used to investigate relationships between c-Met expression, patient characteristics, tumor recurrence, and survival.
c-Met expression was associated with portal vein invasion (p = 0.006). Recurrence-free survival rates were significantly lower in patients with high levels of c-Met expression (p < 0.001). However, c-Met expression levels did not significantly affect overall survival rates (p = 0.12). Only 1 patient was found to have c-Met gene amplification; 22 patients were found to have aneuploidy of chromosome 7, on which the c-Met gene is located. Tumors with chromosome 7 polysomy tended to have higher levels of c-Met expression than those with chromosome 7 monosomy or disomy, but this difference was not statistically significant.
Although c-Met expression was not significantly associated with c-Met gene amplification, it may be a useful predictive marker of recurrence in resected HCC patients.

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Frequent GNAS mutations in low-grade appendiceal mucinous neoplasms

G. Nishikawa, S. Sekine, R. Ogawa, A. Matsubara, T. Mori, H. Taniguchi, R. Kushima, N. Hiraoka, K. Tsuta, H. Tsuda, Y. Kanai

British Journal of Cancer (British Journal of Cancer) 108 ( 4 ) 951 - 958 2013.03

ISSN 00070920

Background:The molecular basis for the development of appendiceal mucinous tumours, which can be a cause of pseudomyxoma peritonei, remains largely unknown.Methods:Thirty-five appendiceal mucinous neoplasms were analysed for GNAS and KRAS mutations. A functional analysis of mutant GNAS was performed using a colorectal cancer cell line.Results:A mutational analysis identified activating GNAS mutations in 16 of 32 low-grade appendiceal mucinous neoplasms (LAMNs) but in none of three mucinous adenocarcinomas (MACs). KRAS mutations were found in 30 LAMNs and in all MACs. We additionally analysed a total of 186 extra-appendiceal mucinous tumours and found that GNAS mutations were highly prevalent in intraductal papillary mucinous tumours of the pancreas (88%) but were rare or absent in mucinous tumours of the colorectum, ovary, lung and breast (0-9%). The prevalence of KRAS mutations was quite variable among the tumours. The introduction of the mutant GNAS into a colorectal cancer cell line markedly induced MUC2 and MUC5AC expression, but did not promote cell growth either in vitro or in vivo.Conclusion:Activating GNAS mutations are a frequent and characteristic genetic abnormality of LAMN. Mutant GNAS might play a direct role in the prominent mucin production that is a hallmark of LAMN. © 2013 Cancer Research UK.

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DNA Methylation Profiles at Precancerous Stages Associated with Recurrence of Lung Adenocarcinoma

Sato, Takashi, Arai, Eri, Kohno, Takashi, Tsuta, Koji, Watanabe, Shun-ichi, Soejima, Kenzo, Betsuyaku, Tomoko, Kanai, Yae

PLOS ONE (PUBLIC LIBRARY SCIENCE) 8 ( 3 ) 798 - 798 2013.03

Research paper (scientific journal), ISSN 1932-6203

The aim of this study was to clarify the significance of DNA methylation alterations at precancerous stages of lung adenocarcinoma. Using single-CpG resolution lnfinium array, genome-wide DNA methylation analysis was performed in 36 samples of normal lung tissue obtained from patients without any primary lung tumor, 145 samples of non-cancerous lung tissue (N) obtained from patients with lung adenocarcinomas, and 145 samples of tumorous tissue (T). Stepwise progression of DNA methylation alterations from normal lung tissue to non-cancerous lung tissue obtained from patients with lung adenocarcinomas, and then tumorous tissue samples, was observed at 3,270 CpG sites, suggesting that non-cancerous lung tissue obtained from patients with lung adenocarcinomas was at precancerous stages with DNA methylation alterations. At CpG sites of 2,083 genes, DNA methylation status in samples of non-cancerous lung tissue obtained from patients with lung adenocarcinomas was significantly correlated with recurrence after establishment of lung adenocarcinomas. Among such recurrence-related genes, 28 genes are normally unmethylated (average beta-values based on lnfinium assay in normal lung tissue samples was less than 0.2) and their DNA hypermethylation at precancerous stages was strengthened during progression to lung adenocarcinomas (Delta beta(T-N)>0.1). Among these 28 genes, we focused on 6 for which implications in transcription regulation, apoptosis or cell adhesion had been reported. DNA hypermethylation of the ADCY5, EVX1, GFRA1, PDE9A, and TBX20 genes resulted in reduced mRNA expression in tumorous tissue samples. 5-Aza-2'-deoxycytidine treatment of lung cancer cell lines restored the mRNA expression levels of these 5 genes. Reduced mRNA expression in tumorous tissue samples was significantly correlated with tumor aggressiveness. These data suggest that DNA methylation alterations at precancerous stages determine tumor aggressiveness and outcome through silencing of specific genes.

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Frequent GNAS and KRAS mutations in pyloric gland adenoma of the stomach and duodenum

Matsubara, Akiko, Sekine, Shigeki, Kushima, Ryoji, Ogawa, Reiko, Taniguchi, Hirokazu, Tsuda, Hitoshi, Kanai, Yae

JOURNAL OF PATHOLOGY (WILEY-BLACKWELL) 229 ( 4 ) 579 - 587 2013.03

Research paper (scientific journal), ISSN 0022-3417

Gastric and duodenal adenomas exhibit a significant morphological and phenotypical diversity and are classified into intestinal-type, foveolar-type and pyloric gland adenomas. We analysed the mutations in GNAS, KRAS, BRAF and CTNNB1 and the expressions of mismatch repair (MMR) proteins in 80 gastric and 32 duodenal adenomas with histologically distinct subtypes, as well as in 71 gastric adenocarcinomas. Activating GNAS mutations were found in 22 of the 35 pyloric gland adenomas (PGAs; 63%) but in none of the foveolar-type or intestinal-type adenomas or the adenocarcinomas. Fourteen PGAs (41%), two foveolar-type adenomas (9%), five intestinal-type adenomas (9%) and one adenocarcinoma (1%) had KRAS mutations. BRAF mutations were absent in all the adenomas and adenocarcinomas that were examined. CTNNB1 mutations were only found in two intestinal-type adenomas (4%). Notably, 13 of the 14 KRAS-mutated gastric and duodenal PGAs had concurrent GNAS mutations. The loss of the MMR proteins, which is indicative of microsatellite instability, was observed in one PGA (3%), 12 foveolar-type adenomas (52%), one intestinal-type adenoma (2%) and five adenocarcinomas (7%). These observations indicate that each histological subtype of gastric and duodenal adenomas has a distinct genetic background. In particular, the present study identified the frequent presence of activating GNAS mutations, which are often associated with KRAS mutations, as a characteristic genetic feature of PGAs of the stomach and duodenum.

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Prevalence of MED12 mutations in uterine and extrauterine smooth muscle tumours

Matsubara, Akiko, Sekine, Shigeki, Yoshida, Masayuki, Yoshida, Akihiko, Taniguchi, Hirokazu, Kushima, Ryoji, Tsuda, Hitoshi, Kanai, Yae

HISTOPATHOLOGY (Histopathology) 62 ( 4 ) 657 - 661 2013.03

Research paper (scientific journal), ISSN 0309-0167

Aims: To determine the prevalence of MED12 mutations in smooth muscle tumours of different organs. Methods and results: A total of 142 smooth muscle tumours of the uterus, gastrointestinal tract, retroperitoneum and soft tissue were analysed for MED12 mutations using Sanger sequencing. Among the uterine tumours that were examined, MED12 mutations were identified in 36 of 45 conventional leiomyomas (80%), two of six cellular leiomyomas (33%), one of four bizarre leiomyomas (25%), none of four lipoleiomyomas (0%), and two of 12 leiomyosarcomas (17%). The two MED12-mutated leiomyosarcomas were associated with benign leiomyomatous components that also harboured MED12 mutations identical to those in the respective leiomyosarcomatous components. None of the extrauterine smooth muscle tumours, including the leiomyomas, leiomyosarcomas, and angioleiomyomas, had MED12 mutations. Conclusions: Among uterine smooth muscle tumours, MED12 mutations are frequently present in conventional leiomyomas, but are significantly less common in histological variants of leiomyoma and leiomyosarcoma. In contrast to uterine lesions, none of the extrauterine smooth muscle tumours had MED12 mutations. © 2012 Blackwell Publishing Ltd.

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3.0-Tesla MRIによる前立腺移行領域癌の診断能とその手術適応について

鳴川司, 中西弘之, 冨田晋太郎, 尾形吉彦, 河原貴史, 原智彦, 込山元清, 新井恵吏, 金井弥栄, 三宅基隆, 眞鍋知子, 藤元博行

日本泌尿器科学会雑誌 ((一社)日本泌尿器科学会) 104 ( 2 ) 358 - 358 2013.03

ISSN 0021-5287

Arginase II Expressed in Cancer-Associated Fibroblasts Indicates Tissue Hypoxia and Predicts Poor Outcome in Patients with Pancreatic Cancer

Ino, Yoshinori, Yamazaki-Itoh, Rie, Oguro, Seiji, Shimada, Kazuaki, Kosuge, Tomoo, Zavada, Jan, Kanai, Yae, Hiraoka, Nobuyoshi

PLOS ONE (PLoS ONE) 8 ( 2 ) 1742 - 1747 2013.02

Research paper (scientific journal), ISSN 1932-6203

An adequate level of arginine in the tissue microenvironment is essential for T cell activity and survival. Arginine levels are regulated by the arginine-catabolizing enzyme, arginase (ARG). It has been reported that arginase II (ARG2), one of two ARGs, is aberrantly expressed in prostate cancer cells, which convert arginine into ornithine, resulting in a lack of arginine that weakens tumor-infiltrating lymphocytes and renders them dysfunctional. However, immune suppression mediated by ARG2-expressing cancer cells in lung cancer has not been observed. Here we studied the expression of ARG2 in pancreatic ductal carcinoma (PDC) tissue clinicopathologically by examining over 200 cases of PDC. In contrast to prostate cancer, ARG2 expression was rarely demonstrated in PDC cells by immunohistochemistry, and instead ARG2 was characteristically expressed in α-smooth muscle actin-positive cancer-associated fibroblasts (CAFs), especially those located within and around necrotic areas in PDC. The presence of ARG2-expressing CAFs was closely correlated with shorter overall survival (OS
P = 0.003) and disease-free survival (DFS
P = 0.0006). Multivariate Cox regression analysis showed that the presence of ARG2-expressing CAFs in PDC tissue was an independent predictor of poorer OS (hazard ratio [HR] = 1.582, P = 0.007) and DFS (HR = 1.715, P = 0.001) in PDC patients. In addition to the characteristic distribution of ARG2-expressing CAFs, such CAFs co-expressed carbonic anhydrase IX, SLC2A1, or HIF-1α, markers of hypoxia, in PDC tissue. Furthermore, in vitro experiments revealed that cultured fibroblasts extracted from PDC tissue expressed the ARG2 transcript after exposure to hypoxia, which had arginase activity. These results indicate that cancer cell-mediated immune suppression through ARG2 expression is not a general event and that the presence of ARG2-expressing CAFs is an indicator of poor prognosis, as well as hypoxia, in PDC tissue. © 2013 Ino et al.

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前立腺癌のADC値と被膜外浸潤の有無との関連についての検討(Can Apparent Diffusion Coefficient (ADC) values contribute to assess the extra capsular extension (ECE) of prostate cancer?)

眞鍋知子, 三宅基隆, 新井恵吏, 金井弥栄, 中西弘之, 込山元清, 藤元博行, 楠本昌彦, 荒井保明

日本医学放射線学会学術集会抄録集 ((公社)日本医学放射線学会) 72回 S369 - S369 2013.02

ISSN 0048-0428

Overexpression of leucine-rich repeat-containing G protein-coupled receptor 5 in gastric cancer

Yamanoi, Kazuhiro, Fukuma, Mariko, Uchida, Hiroshi, Kushima, Ryoji, Yamazaki, Ken, Katai, Hitoshi, Kanai, Yae, Sakamoto, Michiie

PATHOLOGY INTERNATIONAL (Pathology International) 63 ( 1 ) 13 - 19 2013.01

Research paper (scientific journal), ISSN 1320-5463

Gastric cancer is one of the most common malignancies worldwide and patients with advanced gastric cancer still have poor clinical outcomes. The overexpression of leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) mRNA in colorectal cancer and its correlation with clinicopathological factors were recently reported by us. In this study, we show LGR5 mRNA overexpression in human gastric cancer specimens by quantitative RT-PCR and in situ hybridization and assess a correlation with clinicopathological factors. The mean expression of LGR5 mRNA in cancerous tissues was five times higher than that in normal tissue (P=0.0002). Furthermore, LGR5 mRNA expression show marked variation among cases and significantly increased in cases where lymphatic invasion was present compared with those where it was absent (P=0.0056). Although the mean expression level of LGR5 was observed to be higher in nodal metastasis and venous invasion positive cases compared to negative cases, a significant difference was not observed. These results suggest that LGR5 can be a biomarker for malignancy in gastric cancer. © 2012 The Authors. Pathology International © 2012 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd.

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Immune cell infiltration as an indicator of the immune microenvironment of pancreatic cancer.

Ino Y, Yamazaki-Itoh R, Shimada K, Iwasaki M, Kosuge T, Kanai Y, Hiraoka N.

Br J Cancer (NATURE PUBLISHING GROUP) 108 ( 4 ) 914 - 923 2013

Accepted, ISSN 00070920

Background: The host immune reaction is represented by immune/inflammatory cell infiltrates. Here we systematically analysed tumour-infiltrating immune/inflammatory cells in pancreatic ductal carcinoma (PDC) and evaluated their clinicopathological impact.
Methods: Using immunohistochemistry, we examined tumour-infiltrating CD68(+) pan-macrophages, HLA-DR(+)CD68(+) M1 macrophages (M1), CD163(+) or CD204(+) M2 macrophages (M2), CD66b(+) neutrophils (Neu), CD4(+) T cells (CD4(+) T), CD8(+) T cells (CD8(+)T), and FOXP3(+)CD4(+) regulatory T cells (Treg) in 212 cases of PDC, and conducted correlation and survival analyses using the Kaplan Meier method and Cox proportional hazards model.
Results: Higher levels of tumour-infiltrating pan-macrophages, M2, Neu, or the ratio of Tregs to CD4(+)T (%Treg) were significantly associated with shorter survival, whereas higher levels of tumour-infiltrating CD4(+)T, CD8(+)T, or the ratio of M1 to pan-macrophages (%M1) were significantly associated with longer survival. Survival analysis of pairs of these variables revealed that some of the resulting patient groups had exclusively longer survival. We then connected the apparently related factors, and two significant variables emerged: tumour-infiltrating CD4(+)T(high)/CD8(+)T(high)/%Treg(low) and tumour-infiltrating %M1(high)/M2(low). Multivariate survival analysis revealed that these variables were significantly correlated with longer survival and had a higher hazard ratio.
Conclusion: Tumour-infiltrating CD4(+)T(high)/CD8(+)T(high)/%Treg(low) and %M1(high)/M2(low) are independent prognosticators useful for evaluating the immune microenvironment of PDC.

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DNA Methylation Alterations in Human Cancers

Kanai Y., Arai E.

Epigenetics in Human Disease (Epigenetics in Human Disease) 29 - 52 2012.12

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Identification of CCDC6-RET fusion in the human lung adenocarcinoma cell line, LC-2/ad.

Matsubara D, Kanai Y, Ishikawa S, Ohara S, Yoshimoto T, Sakatani T, Oguni S, Tamura T, Kataoka H, Endo S, Murakami Y, Aburatani H, Fukayama M, Niki T

Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 7 ( 12 ) 1872 - 1876 2012.12

Accepted, ISSN 1556-0864

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Characteristics of Lymph Node Metastases Defining the Outcome After Radical Cystectomy of Urothelial Bladder Carcinoma

Nakagawa, Tohru, Kanai, Yae, Nakanishi, Hiroyuki, Komiyama, Motokiyo, Fujimoto, Hiroyuki

JAPANESE JOURNAL OF CLINICAL ONCOLOGY (OXFORD UNIV PRESS) 42 ( 11 ) 1066 - 1072 2012.11

Research paper (scientific journal), ISSN 0368-2811

The aim of this study was to identify clinicopathological variables associated with the clinical outcomes of patients with lymph node metastasis-positive urothelial bladder carcinoma after radical cystectomy.
Forty-six patients who underwent radical cystectomy without preoperative chemotherapy and had histologically proven nodal metastasis were included in the study. The status of lymph nodes and primary lesion was analyzed in terms of disease-specific survival and recurrence-free survival.
The 5-year disease-specific survival and recurrence-free survival for the 46 patients overall were 41.3 and 32.2, respectively. Univariate analysis showed that pN status, the total number of involved lymph nodes, lymph node density and extranodal invasion were statistically significant variables predictive of disease-specific survival. Multivariate analysis revealed that the total number of involved lymph nodes, extranodal invasion and diameter of the metastatic lesion were statistically significant variables predictive of disease-specific survival. Interestingly, the diameter of metastatic lesions was inversely correlated with poorer survival. Patients with large (epsilon 10 mm) metastatic lesions and no extranodal invasion (expansive growth) had significantly better disease-specific survival than those with multiple small (10 mm) metastatic lesions and no extranodal invasion (highly spreading) (P 0.0156) or those with extranodal invasion (infiltrative growth) (P 0.0181).
Our data indicate that the clinical outcome of node-positive patients is not only stratified according to the tumor burden reflected in the total number of involved lymph nodes, but also affected by tumor biology including invasiveness and potential for metastasis, which is reflected in pathological characteristics such as extranodal invasion and the diameter of metastatic lesions.

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Frequent activating GNAS mutations in villous adenoma of the colorectum

Yamada, Masayoshi, Sekine, Shigeki, Ogawa, Reiko, Taniguchi, Hirokazu, Kushima, Ryoji, Tsuda, Hitoshi, Kanai, Yae

JOURNAL OF PATHOLOGY 228 ( 1 ) 113 - 118 2012.09

Research paper (scientific journal), ISSN 0022-3417

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Single-CpG-resolution methylome analysis identifies clinicopathologically aggressive CpG island methylator phenotype clear cell renal cell carcinomas

Arai, Eri, Chiku, Suenori, Mori, Taisuke, Gotoh, Masahiro, Nakagawa, Tohru, Fujimoto, Hiroyuki, Kanai, Yae

CARCINOGENESIS (OXFORD UNIV PRESS) 33 ( 8 ) 1487 - 1493 2012.08

Research paper (scientific journal), ISSN 0143-3334

To clarify the significance of DNA methylation alterations during renal carcinogenesis, methylome analysis using single-CpG-resolution Infinium array was performed on 29 normal renal cortex tissue (C) samples, 107 non-cancerous renal cortex tissue (N) samples obtained from patients with clear cell renal cell carcinomas (RCCs) and 109 tumorous tissue (T) samples. DNA methylation levels at 4830 CpG sites were already altered in N samples compared with C samples. Unsupervised hierarchical clustering analysis based on DNA methylation levels at the 801 CpG sites, where DNA methylation alterations had occurred in N samples and were inherited by and strengthened in T samples, clustered clear cell RCCs into Cluster A (n = 90) and Cluster B (n = 14). Clinicopathologically aggressive tumors were accumulated in Cluster B, and the cancer-free and overall survival rates of patients in this cluster were significantly lower than those of patients in Cluster A. Clear cell RCCs in Cluster B were characterized by accumulation of DNA hypermethylation on CpG islands and considered to be CpG island methylator phenotype (CIMP)-positive cancers. DNA hypermethylation of the CpG sites on the FAM150A, GRM6, ZNF540, ZFP42, ZNF154, RIMS4, PCDHAC1, KHDRBS2, ASCL2, KCNQ1, PRAC, WNT3A, TRH, FAM78A, ZNF671, SLC13A5 and NKX6-2 genes became hallmarks of CIMP in RCCs. On the other hand, Cluster A was characterized by genome-wide DNA hypomethylation. These data indicated that DNA methylation alterations at precancerous stages may determine tumor aggressiveness and patient outcome. Accumulation of DNA hypermethylation on CpG islands and genome-wide DNA hypomethylation may each underlie distinct pathways of renal carcinogenesis.

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Fenton Reaction Induced Cancer in Wild Type Rats Recapitulates Genomic Alterations Observed in Human Cancer

Akatsuka, Shinya, Yamashita, Yoriko, Ohara, Hiroki, Liu, Yu-Ting, Izumiya, Masashi, Abe, Koichiro, Ochiai, Masako, Jiang, Li, Nagai, Hirotaka, Okazaki, Yasumasa, Murakami, Hideki, Sekido, Yoshitaka, Arai, Eri, Kanai, Yae, Hino, Okio, Takahashi, Takashi, Nakagama, Hitoshi, Toyokuni, Shinya

PLOS ONE (PUBLIC LIBRARY SCIENCE) 7 ( 8 ) 207 - 213 2012.08

Research paper (scientific journal), ISSN 1932-6203

Iron overload has been associated with carcinogenesis in humans. Intraperitoneal administration of ferric nitrilotriacetate initiates a Fenton reaction in renal proximal tubules of rodents that ultimately leads to a high incidence of renal cell carcinoma (RCC) after repeated treatments. We performed high-resolution microarray comparative genomic hybridization to identify characteristics in the genomic profiles of this oxidative stress-induced rat RCCs. The results revealed extensive large-scale genomic alterations with a preference for deletions. Deletions and amplifications were numerous and sometimes fragmented, demonstrating that a Fenton reaction is a cause of such genomic alterations in vivo. Frequency plotting indicated that two of the most commonly altered loci corresponded to a Cdkn2a/2b deletion and a Met amplification. Tumor sizes were proportionally associated with Met expression and/or amplification, and clustering analysis confirmed our results. Furthermore, we developed a procedure to compare whole genomic patterns of the copy number alterations among different species based on chromosomal syntenic relationship. Patterns of the rat RCCs showed the strongest similarity to the human RCCs among five types of human cancers, followed by human malignant mesothelioma, an iron overload-associated cancer. Therefore, an iron-dependent Fenton chemical reaction causes large-scale genomic alterations during carcinogenesis, which may result in distinct genomic profiles. Based on the characteristics of extensive genome alterations in human cancer, our results suggest that this chemical reaction may play a major role during human carcinogenesis.

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Expression of a splicing variant of the CADM1 specific to small cell lung cancer

Kikuchi, Shinji, Iwai, Miwako, Sakurai-Yageta, Mika, Tsuboi, Yumi, Ito, Takeshi, Maruyama, Tomoko, Tsuda, Hitoshi, Kanai, Yae, Onizuka, Masataka, Sato, Yukio, Murakami, Yoshinori

CANCER SCIENCE (WILEY-BLACKWELL) 103 ( 6 ) 1051 - 1057 2012.06

Research paper (scientific journal), ISSN 1347-9032

CADM1, a member of the immunoglobulin superfamily cell adhesion molecule, acts as a tumor suppressor in a variety of human cancers. CADM1 is also ectopically expressed in adult T-cell leukemia (ATL), conferring an invasive phenotype characteristic to ATL. Therefore, CADM1 plays dual roles in human oncogenesis. Here, we investigate the roles of CADM1 in small cell lung cancer (SCLC). Immunohistochemistry demonstrates that 10 of 35 (29%) primary SCLC tumors express CADM1 protein. Western blotting and RT-PCR analyses reveal that CADM1 is significantly expressed in 11 of 14 SCLC cells growing in suspension cultures but in neither of 2 SCLC cells showing attached growth to plastic dishes, suggesting that CADM1 is involved in anchorage-independent growth in SCLC. In the present study, we demonstrate that SCLC expresses a unique splicing variant of CADM1 (variant 8/9) containing additional extracellular fragments corresponding to exon 9 in addition to variant 8, a common isoform in epithelia. Variant 8/9 of CADM1 is almost exclusively observed in SCLC and testis, although this variant protein localizes along the membrane and shows similar cell aggregation activity to variant 8. Interestingly, both variant 8/9 and variant 8 of CADM1 show enhanced tumorigenicity in nude mice when transfected into SBC5, a SCLC cell lacking CADM1. Inversely, suppression of CADM1 expression by shRNA reduced spheroid-like cell aggregation of NCI-H69, an SCLC cell expressing a high amount of CADM1. These findings suggest that CADM1 enhances the malignant features of SCLC, as is observed in ATL, and could provide a molecular marker specific to SCLC. (Cancer Sci 2012; 103: 10511057)

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Potential role of LMP2 as an anti-oncogenic factor in human uterine leiomyosarcoma: Morphological significance of calponin h1

Hayashi, Takuma, Horiuchi, Akiko, Sano, Kenji, Hiraoka, Nobuyoshi, Kasai, Mari, Ichimura, Tomoyuki, Sudo, Tamotsu, Nishimura, Ryuichiro, Ishiko, Osamu, Shiozawa, Tanri, Kanai, Yae, Yaegashi, Nobuo, Aburatani, Hiroyuki, Konishi, Ikuo

FEBS LETTERS (ELSEVIER SCIENCE BV) 586 ( 13 ) 1824 - 1831 2012.06

Research paper (scientific journal), ISSN 0014-5793

Uterine leiomyosarcoma (LMS) is a highly metastatic smooth muscle neoplasm for which calponin h1 is suspected to have a biological role as a tumor-suppressor. We earlier reported that LMP2-null mice spontaneously develop uterine LMS through malignant transformation of the myometrium, thus implicating this protein as an anti-tumorigenic candidate as well. In the present study, we show that LMP2 may negatively regulate LMS independently of its role in the proteasome. Moreover, several lines of evidence indicate that although calponin h1 does not directly influence tumorigenesis, it clearly affects LMP2-induced cellular morphological changes. Modulation of LMP2 may lead to new therapeutic approaches in human uterine LMS. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

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DNA methyltransferase 3B expression is associated with poor outcome of stage I testicular seminoma

Arai, Eri, Nakagawa, Tohru, Wakai-Ushijima, Saori, Fujimoto, Hiroyuki, Kanai, Yae

HISTOPATHOLOGY (WILEY-BLACKWELL) 60 ( 6B ) E12 - E18 2012.05

Research paper (scientific journal), ISSN 0309-0167

Aims: To examine in testicular seminomas the expression of DNA methyltransferase 3B (DNMT3B), which is known to be associated with early embryonic development and carcinogenesis, and to obtain a predictive marker for relapse of stage I seminomas.
Methods and results: Immunohistochemical examination of DNMT3B was performed in 88 cases of seminoma, 35 (39.8%) of which showed widely scattered nuclear immunoreactivity for DNMT3B, and 53 (60.2%) of which were completely negative. The incidence of focal DNMT3B expression was higher in stage III seminomas (5/5, 100%) than in stage I (25/70, 35.7%) or stage II (5/13, 38.5%) seminomas (P = 0.011). In stage I seminomas there were no significant correlations between DNMT3B expression and tumour size, invasion of the rete testis, or lymphatic or vascular involvement. Six of 25 cases (24%) showing DNMT3B expression relapsed, whereas only 3/45 cases (6.7%) lacking such expression did so (P = 0.037). Patients with seminomas showing DNMT3B expression had a significantly lower relapse-free survival rate than patients whose tumours lacked this feature (P = 0.0464).
Conclusions: Patients with seminomas showing focal DNMT3B expression are at increased risk of relapse, and should be followed up carefully.

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Morules in Intraductal Papillary Mucinous Neoplasm With an Associated Invasive Carcinoma of the Pancreas

Tateno, Yoko, Esaki, Minoru, Shimada, Kazuaki, Ojima, Hidenori, Kanai, Yae, Hiraoka, Nobuyoshi

PANCREAS 41 ( 4 ) 651 - 652 2012.05

Research paper (scientific journal), ISSN 0885-3177

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Higher-Order Chromatin Regulation and Differential Gene Expression in the Human Tumor Necrosis Factor/Lymphotoxin Locus in Hepatocellular Carcinoma Cells

Watanabe, Takehisa, Ishihara, Ko, Hirosue, Akiyuki, Watanabe, Sugiko, Hino, Shinjiro, Ojima, Hidenori, Kanai, Yae, Sasaki, Yutaka, Nakao, Mitsuyoshi

MOLECULAR AND CELLULAR BIOLOGY (AMER SOC MICROBIOLOGY) 32 ( 8 ) 1529 - 1541 2012.04

Research paper (scientific journal), ISSN 0270-7306

The three-dimensional context of endogenous chromosomal regions may contribute to the regulation of gene clusters by influencing interactions between transcriptional regulatory elements. In this study, we investigated the effects of tumor necrosis factor (TNF) signaling on spatiotemporal enhancer-promoter interactions in the human tumor necrosis factor (TNF)/lymphotoxin (LT) gene locus, mediated by CCCTC-binding factor (CTCF)-dependent chromatin insulators. The cytokine genes LT alpha, TNF, and LT beta are differentially regulated by NF-kappa B signaling in inflammatory and oncogenic responses. We identified at least four CTCF-enriched sites with enhancer-blocking activities and a TNF-responsive TE2 enhancer in the TNF/LT locus. One of the CTCF-enriched sites is located between the early-inducible LT alpha/TNF promoters and the late-inducible LT beta promoter. Depletion of CTCF reduced TNF expression and accelerated LT beta induction. After TNF stimulation, via intrachromosomal dynamics, these insulators mediated interactions between the enhancer and the LT alpha/TNF promoters, followed by interaction with the LT beta promoter. These results suggest that insulators mediate the spatiotemporal control of enhancer-promoter associations in the TNF/LT gene cluster.

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Whole-exome sequencing of human pancreatic cancers and characterization of genomic instability caused by MLH1 haploinsufficiency and complete deficiency

Wang, Linghua, Tsutsumi, Shuichi, Kawaguchi, Tokuichi, Nagasaki, Koichi, Tatsuno, Kenji, Yamamoto, Shogo, Sang, Fei, Sonoda, Kohtaro, Sugawara, Minoru, Saiura, Akio, Hirono, Seiko, Yamaue, Hiroki, Miki, Yoshio, Isomura, Minoru, Totoki, Yasushi, Nagae, Genta, Isagawa, Takayuki, Ueda, Hiroki, Murayama-Hosokawa, Satsuki, Shibata, Tatsuhiro, Sakamoto, Hiromi, Kanai, Yae, Kaneda, Atsushi, Noda, Tetsuo, Aburatani, Hiroyuki

GENOME RESEARCH (COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT) 22 ( 2 ) 208 - 219 2012.02

Research paper (scientific journal), ISSN 1088-9051

Whole-exome sequencing (Exome-seq) has been successfully applied in several recent studies. We here sequenced the exomes of 15 pancreatic tumor cell lines and their matched normal samples. We captured 162,073 exons of 16,954 genes and sequenced the targeted regions to a mean coverage of 56-fold. This study identified a total of 1517 somatic mutations and validated 934 mutations by transcriptome sequencing. We detected recurrent mutations in 56 genes. Among them, 41 have not been described. The mutation rates varied widely among cell lines. The diversity of the mutation rates was significantly correlated with the distinct MLH1 copy-number status. Exome-seq revealed intensive genomic instability in a cell line with MLH1 homozygous deletion, indicated by a dramatically elevated rate of somatic substitutions, small insertions/deletions (indels), as well as indels in microsatellites. Notably, we found that MLH1 expression was decreased by nearly half in cell lines with an allelic loss of MLH1. While these cell lines were negative in conventional microsatellite instability assay, they showed a 10.5-fold increase in the rate of somatic indels, e.g., truncating indels in TP53 and TGFBR2, indicating MLH1 haploinsufficiency in the correction of DNA indel errors. We further analyzed the exomes of 15 renal cell carcinomas and confirmed MLH1 haploinsufficiency. We observed a much higher rate of indel mutations in the affected cases and identified recurrent truncating indels in several cancer genes such as VHL, PBRM1, and JARIDIC. Together, our data suggest that MLH1 hemizygous deletion, through increasing the rate of indel mutations, could drive the development and progression of sporadic cancers.

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Prostate Stem Cell Antigen, a Presumable Organ-Dependent Tumor Suppressor Gene, is Down-Regulated in Gallbladder Carcinogenesis

Ono, Hiroe, Hiraoka, Nobuyoshi, Lee, Yeon-Su, Woo, Sang Myung, Lee, Woo Jin, Choi, Il Ju, Saito, Akira, Yanagihara, Kazuyoshi, Kanai, Yae, Ohnami, Sumiko, Chiwaki, Fumiko, Sasaki, Hiroki, Sakamoto, Hiromi, Yoshida, Teruhiko, Saeki, Norihisa

GENES CHROMOSOMES & CANCER (WILEY) 51 ( 1 ) 30 - 41 2012.01

Research paper (scientific journal), ISSN 1045-2257

Gallbladder cancer (GBC) is relatively rare but has a high mortality rate. One candidate molecule which might be involved in GBC development is prostate stem cell antigen (PSCA), a glycosylphosphatidylinositol- anchored cell surface antigen with a tissue- specific pattern of expression in the epithelium of several organs, such as the prostate, stomach, bladder, and gallbladder. It is up- regulated in a number of cancers including prostate, urinary bladder, and pancreatic cancers, while it is down- regulated in esophageal and gastric cancers, suggesting that PSCA has an oncogenic activity in the former but a tumor suppressor activity in the latter. However, the precise function of PSCA and the regulatory mechanism for its expression in normal and cancer cells are yet to be determined. In this study, immunohistochemical analyses with a specific antibody revealed that PSCA is down- regulated in non- neoplastic gallbladder lesions such as cholesterolosis, cholecystolithiasis, and cholecystitis (9/17; 53%), and also in adenocarcinoma (40/44; 91%), a common neoplasm in gallbladder. Analyses of the DNA methylation status in the GBC cell lines by bisulfite- Pyrosequencing and a reporter assay for the PSCA promoter activity suggested that the down- regulation is explained, at least partly, by DNA methylation. Moreover, colony formation assay revealed that PSCA has cell- proliferation inhibition activity in the GBC cell lines, which was also observed in vivo. These lines of in vivo and in vitro evidence suggest that PSCA is acting as a tumor suppressor in GBC development. (C) 2011 Wiley Periodicals, Inc.

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Frequent activating GNAS mutations in villous adenoma of the colorectum.

Yamada M, Sekine S, Ogawa R, Taniguchi H, Kushima R, Tsuda H, Kanai Y.

J Pathol 228 113 - 118 2012

Accepted

Potential role of LMP2 as tumor-suppressor defines new targets for uterine leiomyosarcoma therapy

Hayashi, Takuma, Horiuchi, Akiko, Sano, Kenji, Hiraoka, Nobuyoshi, Kasai, Mari, Ichimura, Tomoyuki, Sudo, Tamotsu, Tagawa, Yoh-ichi, Nishimura, Ryuichiro, Ishiko, Osamu, Kanai, Yae, Yaegashi, Nobuo, Aburatani, Hiroyuki, Shiozawa, Tanri, Konishi, Ikuo

SCIENTIFIC REPORTS (NATURE PUBLISHING GROUP) 1 907 - 912 2011.12

Research paper (scientific journal), ISSN 2045-2322

Although the majority of smooth muscle neoplasms found in the uterus are benign, uterine leiomyosarcoma (LMS) is extremely malignant, with high rates of recurrence and metastasis. We earlier reported that mice with a homozygous deficiency for LMP2, an interferon (IFN)-gamma-inducible factor, spontaneously develop uterine LMS. The IFN-gamma pathway is important for control of tumor growth and invasion and has been implicated in several cancers. In this study, experiments with human and mouse uterine tissues revealed a defective LMP2 expression in human uterine LMS that was traced to the IFN-gamma pathway and the specific effect of JAK-1 somatic mutations on the LMP2 transcriptional activation. Furthermore, analysis of a human uterine LMS cell line clarified the biological significance of LMP2 in malignant myometrium transformation and cell cycle, thus implicating LMP2 as an anti-tumorigenic candidate. This role of LMP2 as a tumor suppressor may lead to new therapeutic targets in human uterine LMS.

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Carcinogenetic risk estimation based on quantification of DNA methylation levels in liver tissue at the precancerous stage

Nagashio, Ryo, Arai, Eri, Ojima, Hidenori, Kosuge, Tomoo, Kondo, Yutaka, Kanai, Yae

INTERNATIONAL JOURNAL OF CANCER (WILEY-BLACKWELL) 129 ( 5 ) 1170 - 1179 2011.09

Research paper (scientific journal), ISSN 0020-7136

For appropriate surveillance of patients at the precancerous stage for hepatocellular carcinomas (HCCs), carcinogenetic risk estimation is advantageous. The aim of our study was to establish criteria for such estimation based on DNA methylation profiling. The DNA methylation status of 203 CpG sites on 25 bacterial artificial chromosome (BAC) clones, whose DNA methylation status had been proven to discriminate samples of noncancerous liver tissue obtained from patients with HCC (N) from normal liver tissue (C) samples by BAC array-based methylated CpG island amplification, was evaluated quantitatively using pyrosequencing. The 45 CpG sites whose DNA methylation levels differed significantly between C and N in the learning cohort (n = 22) were identified. The criteria combining DNA methylation status for the 30 regions including the 45 CpG sites were able to diagnose N as being at high risk of carcinogenesis with 100% sensitivity and specificity in the learning cohort and 95.6% sensitivity and 100% specificity in the validation (n = 90) cohort. DNA methylation status for the 30 regions in N samples was significantly correlated with the outcome of patients with HCCs, indicating that clinicopathologically valid DNA methylation alterations have already accumulated at the precancerous stage. The DNA methylation status of the 30 regions did not depend on the presence or absence of hepatitis virus infection, or the status of noncancerous liver tissue (chronic hepatitis or cirrhosis). These criteria may be applicable for carcinogenetic risk estimation using liver biopsy specimens obtained from patients who are followed up because of chronic liver diseases.

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Expression of SLCO1B3 is associated with intratumoral cholestasis and CTNNB1 mutations in hepatocellular carcinoma

Sekine, Shigeki, Ogawa, Reiko, Ojima, Hidenori, Kanai, Yae

CANCER SCIENCE (WILEY-BLACKWELL) 102 ( 9 ) 1742 - 1747 2011.09

Research paper (scientific journal), ISSN 1347-9032

Recent studies have shown that intratumoral cholestasis is a hallmark of CTNNB1 mutations in hepatocellular carcinomas (HCC). Here, we analyzed the expressions of genes involved in bile acid and bilirubin metabolism and their correlation with the mutational status of CTNNB1 in a series of HCC. The expressions of CYP7A1 and CYP27A1, which encode rate-limiting enzymes in bile acid synthesis, were unaltered or only marginally increased in CTNNB1-mutated HCC compared with those in HCC with wild-type CTNNB1. Among the genes involved in bile acid and bilirubin transport, the expression of SLCO1B3 was significantly elevated in HCC with CTNNB1 mutations, whereas the expression of ABCC4 was elevated in HCC with wild-type CTNNB1. Immunohistochemistry confirmed the frequent expression of SLCO1B3 in CTNNB1-mutated HCC at the protein level, but not in most HCC with wild-type CTNNB1. Immunohistochemistry for MRP4 (encoded by ABCC4) partly agreed with ABCC4 expression, but most cases did not express detectable levels of MRP4. Notably, all HCC with bile accumulation, including those without CTNNB1 mutations, expressed SLCO1B3, suggesting that SLCO1B3 expression, rather than CTNNB1 mutation, is the critical determinant of intratumoral cholestasis. As SLCO1B3 is involved in the uptake of a number of chemotherapeutic and diagnostic agents, SLCO1B3 expression and the status of CTNNB1 mutation might need to be considered in the drug delivery to HCC. (Cancer Sci 2011; 102: 1742-1747)

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Copy number alterations in urothelial carcinomas: their clinicopathological significance and correlation with DNA methylation alterations

Nishiyama, Naotaka, Arai, Eri, Nagashio, Ryo, Fujimoto, Hiroyuki, Hosoda, Fumie, Shibata, Tatsuhiro, Tsukamoto, Taiji, Yokoi, Sana, Imoto, Issei, Inazawa, Johji, Kanai, Yae

CARCINOGENESIS (OXFORD UNIV PRESS) 32 ( 4 ) 462 - 469 2011.04

Research paper (scientific journal), ISSN 0143-3334

The aim of this study was to clarify the genetic backgrounds underlying the clinicopathological characteristics of urothelial carcinomas (UCs). Array comparative genomic hybridization analysis using a 244K oligonucleotide array was performed on 49 samples of UC tissue. Losses of 2q33.3-q37.3, 4p15.2-q13.1 and 5q13.3-q35.3 and gains of 7p11.2-q11.23 and 20q13.12-q13.2 were correlated with higher histological grade, and gain of 7p21.2-p21.12 was correlated with deeper invasion. Losses of 6q14.1-q27 and 17p13.3-q11.1 and gains of 19q13.12-q13.2 and 20q13.12-q13.33 were correlated with lymph vessel involvement. Loss of 16p12.2-p12.1 and gain of 3q26.32-q29 were correlated with vascular involvement. Losses of 5q14.1-q23.1, 6q14.1-q27, 8p22-p21.3, 11q13.5-q14.1 and 15q11.2-q22.2 and gains of 7p11.2-q11.22 and 19q13.12-q13.2 were correlated with the development of aggressive non-papillary UCs. Losses of 1p32.2-p31.3, 10q11.23-q21.1 and 15q21.3 were correlated with tumor recurrence. Unsupervised hierarchical clustering analysis based on copy number alterations clustered UCs into three subclasses: copy number alterations associated with genome-wide DNA hypomethylation, regional DNA hypermethylation on C-type CpG islands and genome-wide DNA hypo- and hypermethylation were accumulated in clusters A, B(1) and B(2), respectively. Tumor-related genes that may encode therapeutic targets and/or indicators useful for the diagnosis and prognostication of UCs should be explored in the above regions. Both genetic and epigenetic events appear to accumulate during urothelial carcinogenesis, reflecting the clinicopathological diversity of UCs.

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Hepatomas with activating Ctnnb1 mutations in 'Ctnnb1-deficient' livers: a tricky aspect of a conditional knockout mouse model

Sekine, Shigeki, Ogawa, Reiko, Kanai, Yae

CARCINOGENESIS (OXFORD UNIV PRESS) 32 ( 4 ) 622 - 628 2011.04

Research paper (scientific journal), ISSN 0143-3334

Conditional knockout mice, based on the Cre-loxP system, are a widely used model for examining organ-specific gene functions. To date, efficient hepatocyte-specific knockout has been reported in many different models, but little attention has been paid to the long-term stability of the recombination efficiency. In the present study, we characterized Alb-Cre;Ctnnb1(flox/flox) 'hepatocyte-specific Ctnnb1 knockout' mice of different ages to test whether efficient recombination is maintained over time. At 2 months of age, the knockout mouse livers achieved efficient deletions of beta-catenin in hepatocytes. However, as the mice aged, the reappearance and expansion of beta-catenin-expressing hepatocytes were observed. In 1-year-old mice, a significant proportion of the pericentral hepatocytes in the knockout mouse livers were replaced with beta-catenin-positive hepatocytes, whereas the periportal hepatocytes mostly remained beta-catenin-negative. Furthermore, most of the 1-year-old mice spontaneously developed hepatocellular adenomas and carcinomas that were positive for beta-catenin and overexpressed glutamine synthetase and Slc1a2, both of which are hallmarks of active beta-catenin signaling. Sequencing analysis revealed that the Ctnnb1 alleles were not inactivated but had activating mutations in these tumors. The present study suggests that recombination efficiency should be carefully examined when hepatocyte-specific knockout mice of different ages are analyzed. In addition, illegitimate deletion mutations should be recognized as potential adverse effects of the Cre-loxP system.

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Overexpression of alpha-methylacyl-CoA racemase is associated with CTNNB1 mutations in hepatocellular carcinomas

Sekine, Shigeki, Ogawa, Reiko, Ojima, Hidenori, Kanai, Yae

HISTOPATHOLOGY (WILEY-BLACKWELL) 58 ( 5 ) 712 - 719 2011.04

Research paper (scientific journal), ISSN 0309-0167

Aims:
alpha-Methylacyl-CoA racemase (AMACR) is expressed in the majority of hepatocellular carcinomas (HCCs) at variable levels, but the significance of AMACR overexpression remains elusive. The aim of this study was to investigate the relationship between AMACR expression and the presence of CTNNB1 mutations in HCCs.
Methods and results:
The expression of AMACR and GLUL, an established downstream target of beta-catenin was examined in HCCs, by quantitative reverse transcription polymerase chain reaction (PCR), and the expression of their protein products by immunohistochemistry. The quantitative reverse transcription PCR analysis showed that the expression of AMACR was significantly higher in HCCs with CTNNB1 mutations than in mutation-negative HCCs or normal livers, like the expression of GLUL. Immunohistochemistry also showed that strong AMACR protein expression was closely correlated with the presence of CTNNB1 mutations. HCCs with CTNNB1 mutations and those with AMACR overexpression frequently exhibited bile production.
Conclusions:
The overexpression of AMACR was closely correlated with the presence of CTNNB1 mutations in HCCs. AMACR is a putative target of beta-catenin as well as an excellent immunohistochemically detectable marker of HCCs with CTNNB1 mutations. As AMACR is physiologically involved in bile acid synthesis, the current observation implies a regulatory role of beta-catenin in bile acid metabolism.

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CXCL17 and ICAM2 Are Associated With a Potential Anti-Tumor Immune Response in Early Intraepithelial Stages of Human Pancreatic Carcinogenesis

Hiraoka, Nobuyoshi, Yamazaki-Itoh, Rie, Ino, Yoshinori, Mizuguchi, Yasunori, Yamada, Tesshi, Hirohashi, Setsuo, Kanai, Yae

GASTROENTEROLOGY (W B SAUNDERS CO-ELSEVIER INC) 140 ( 1 ) 310 - + 2011.01

Research paper (scientific journal), ISSN 0016-5085

BACKGROUND & AIMS: Anti-tumor immunity changes over the course of tumor progression; it is not clear how or when the developing tumor overcomes immune surveillance. Intraductal papillary mucinous neoplasm (IPMN) is an intraepithelial precursor lesion of pancreatic cancer that progresses from adenoma to carcinoma. We investigated when and how the human anti-tumor immune reaction changes during pancreatic tumor development. METHODS: Using immunohistochemical analysis of cells isolated from patients with IPMN, the numbers of tumor-infiltrating lymphocytes and dendritic cells and the maturation state of dendritic cells in the regional lymph nodes were investigated during tumor progression. Gene expression profiles were compared among epithelial neoplastic cells at each stage of tumor development. Biological functions of the selected gene products were analyzed using syngeneic mouse models. RESULTS: The antitumor immune reaction changed from an immune response to immune tolerance between the stages of intraductal papillary mucinous adenoma (IPMA) and intraductal papillary mucinous carcinoma (IPMC). Chemokine (C-X-C motif) ligand 17 (CXCL17) and intercellular adhesion molecule 2 (ICAM2) were involved in immune surveillance during tumor development-their expression levels were up-regulated exclusively in IPMA and disappeared from IPMC. CXCL17 and ICAM2 induced infiltration and accumulation of the tumor epithelial layer by immature myeloid dendritic cells. This was followed by a cellular immune reaction and ICAM2 simultaneously promoted the susceptibility of the tumor cells to cytotoxic T-cell-mediated cytolysis. These processes had a synergistic effect to increase the anti-tumor immune response. CONCLUSIONS: Immune surveillance occurs during the early intraepithelial stages of human pancreatic carcinogenesis and is mediated by expression of CXCL17 and ICAM2.

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Genome-Wide DNA Methylation Profiles in Renal Tumors of Various Histological Subtypes and Non-Tumorous Renal Tissues

Arai, Eri, Wakai-Ushijima, Saori, Fujimoto, Hiroyuki, Hosoda, Fumie, Shibata, Tatsuhiro, Kondo, Tadashi, Yokoi, Sana, Imoto, Issei, Inazawa, Johji, Hirohashi, Setsuo, Kanai, Yae

PATHOBIOLOGY (KARGER) 78 ( 1 ) 1 - 9 2011

Research paper (scientific journal), ISSN 1015-2008

Objective: The aim of this study is to clarify genome-wide DNA methylation profiles in renal tumors of various histological subtypes. Methods: Bacterial artificial chromosome (BAC) array-based methylated CpG island amplification was performed using tissue samples of 17 patients with papillary renal cell carcinomas (RCCs), chromophobe RCCs and oncocytomas, and the results were compared with those from 51 patients with clear cell RCCs. Results: Unsupervised hierarchical clustering analysis based on DNA methylation status clustered type 1 and type 2 papillary RCCs into different subclasses. Although chromophobe RCCs and oncocytomas were clustered into the same subclass, the DNA methylation status of 21 BAC clones was able to discriminate chromophobe RCCs from oncocytomas. The number of BAC clones showing DNA methylation alteration in non-tumorous renal tissue from patients with chronnophobe RCCs and oncocytomas was smaller than that from patients with clear cell RCCs. Biphasic accumulation of DNA methylation alterations was observed in non-tumorous renal tissue from all 68 patients, and patients showing such alterations on more BAC clones had a poorer outcome than patients showing them on fewer BAC clones. Conclusions: DNA methylation profiles determining the histological subtypes of renal tumors developing in individual patients and/or patient outcome may be already established in non-tumorous renal tissue at the precancerous stage. Copyright (c) 2011 S. Karger AG, Basel

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Involvement of proteasome β1i subunit, LMP2, on development of uterin leiomyosarcma.

Hayashi T, Horiuchi A, Sano K, Hiraoka N, Kasai M, Ichimura T, Nagase S, Ishiko O, Shiozawa T, Kanai Y, Yaegashi N, Aburatani H, Tonegawa S, Konishi I.

N Am J Med Sci (North American Journal of Medical Sciences) 3 ( 9 ) 394 - 399 2011

Accepted, ISSN 1947-2714

Background: Although the majority of smooth muscle neoplasms found in the uterus are benign, uterine leiomyosarcoma is extremely malignant, with high rates of recurrence and metastasis. The development of gynecologic tumors is often correlated with secretion of female hormone
however, the development of human uterine leiomyosarcoma is not substantially correlated with hormonal conditions, and the risk factors are unclearly understood. Importantly, a diagnostic-biomarker, which distinguishes malignant human uterine leiomyosarcoma from benign tumor leiomyoma is yet to be established. Aims: It is necessary to analyze risk factors associated with human uterine leiomyosarcoma, in order to establish a diagnostic-biomarker and a clinical treatment method. Patients and Methods: Histology and Immunofluorescence Staining: Uteri obtained from LMP2-/- mice or its parental mice (C57BL/6 mice) were fixed in 10% buffered formalin, incubated in 4% paraformaldehyde for 8 hours, and embedded in paraffin. Tissue sections (5 μm) were prepared and stained with H&amp
E for routine histological examination or were processed further for immunofluorescence staining with appropriate antidodies. Furthermore, a total of 101 patients between 32 and 83 years of age and diagnosed as having smooth muscle tumors of the uterus were selected from pathological files. Immunohistochemistry staining for LMP2 was performed on serial human uterine leiomyosarcoma, leiomyoma and myometrium sections. Results: Homozygous deficient mice for a proteasome β1i subunit, LMP2 spontaneously develop uterine leiomyosarcoma, with a disease prevalence of ~40% by 14 months of age. Defective LMP2 expression in human uterine leiomyosarcoma was demonstrated, but present in human leiomyoma and myometrium. Conclusions: Loss in LMP2 expression may be one of the risk factors for human uterine leiomyosarcoma. LMP2 may be a potential diagnostic-biomarker and targeted-molecule for a new therapeutic approach.

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Molecular Approach to Uterine Leiomyosarcoma: LMP2-Deficient Mice as an Animal Model of Spontaneous Uterine Leiomyosarcoma.

Hayashi T, Horiuchi A, Sano K, Hiraoka N, Kanai Y, Shiozawa T, Tonegawa S, Konishi I.

Sarcoma (Sarcoma) 2011 476498 2011

Accepted, ISSN 1357714X

Uterine leiomyosarcoma (LMS) develops more often in the muscle tissue layer of the uterine body than in the uterine cervix. The development of gynecologic tumors is often correlated with female hormone secretion
however, the development of uterine LMS is not substantially correlated with hormonal conditions, and the risk factors are not yet known. Importantly, a diagnostic-biomarker which distinguishes malignant LMS from benign tumor leiomyoma (LMA) is yet to be established. Accordingly, it is necessary to analyze risk factors associated with uterine LMS, in order to establish a treatment method. LMP2-deficient mice spontaneously develop uterine LMS, with a disease prevalence of ~40 by 14 months of age. We found LMP2 expression to be absent in human LMS, but present in human LMA. Therefore, defective LMP2 expression may be one of the risk factors for LMS. LMP2 is a potential diagnostic-biomarker for uterine LMS, and may be targeted-molecule for a new therapeutic approach. Copyright © 2011 Takuma Hayashi et al.

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Use of quantitative shotgun proteomics to identify fibronectin 1 as a potential plasma biomarker for clear cell carcinoma of the kidney

Yokomizo, Akira, Takakura, Michiko, Kanai, Yae, Sakuma, Tomohiro, Matsubara, Junichi, Honda, Kazufumi, Naito, Seiji, Yamada, Tesshi, Ono, Masaya

CANCER BIOMARKERS (IOS PRESS) 10 ( 3-4 ) 175 - 183 2011

Research paper (scientific journal), ISSN 1574-0153

Background: Early detection would be one of the most effective means to improve the outcome of renal cell carcinoma (RCC). We searched for a new plasma marker for RCC using a label-free quantitative shotgun proteomics method.
Methods: Plasma proteins were digested by trypsin, and the resulting peptides were analyzed by 2-Dimensional Image Converted Analysis of Liquid chromatography mass spectrometry (2DICAL). An identified biomarker candidate was subjected to validation using the Amplified Luminescent Proximity Homogeneous Assay (AlphaLISA).
Results: Among a total of 23,407 independent MS peaks, we found that the mean intensity of 59 peaks significantly differed between 20 clear cell RCC patients and 20 healthy controls. MS/MS spectra from 16 of the 59 peaks matched the amino acid sequences of the fibronectin 1 (FN1) gene product. The increased plasma level of FN1 in RCC patients was validated in a cohort of in 77 patients and 130 healthy controls (p < 0.0001).
Conclusions: The FN1 is considered to be a promising biomarker candidate for clear cell RCC. Furthermore, AlphaLISA is an alternate to the conventional enzyme-linked immunosorbent assay and should prove useful for the rapid validation of biomarker candidates.

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ARAP3 inhibits peritoneal dissemination of scirrhous gastric carcinoma cells by regulating cell adhesion and invasion.

Yagi R, Tanaka M, Sasaki K, Kamata R, Nakanishi Y, Kanai Y, Sakai R.

Oncogene (NATURE PUBLISHING GROUP) 30 ( 12 ) 1413 - 1421 2011

Accepted, ISSN 09509232

During the analysis of phosphotyrosine-containing proteins in scirrhous gastric carcinoma cell lines, we observed an unusual expression of Arf-GAP with Rho-GAP domain, ankyrin repeat and PH domain 3 (ARAP3), a multimodular signaling protein that is a substrate of Src family kinases. Unlike other phosphotyrosine proteins, such as CUB domain-containing protein 1 (CDCP1) and Homo sapiens chromosome 9 open reading frame 10/oxidative stress-associated Src activator (C9orf10/Ossa), which are overexpressed and hyperphosphorylated in scirrhous gastric carcinoma cell lines, ARAP3 was underexpressed in cancerous human gastric tissues. In this study, we found that overexpression of ARAP3 in the scirrhous gastric carcinoma cell lines significantly reduced peritoneal dissemination. In vitro studies also showed that ARAP3 regulated cell attachment to the extracellular matrix, as well as invasive activities. These effects were suppressed by mutations in the Rho-GTPase-activating protein (GAP) domain or in the C-terminal two tyrosine residues that are phosphorylated by Src. Thus, the expression and phosphorylation state of ARAP3 may affect the invasiveness of cancer by modulating cell adhesion and motility. Our results suggest that ARAP3 is a unique Src substrate that suppresses peritoneal dissemination of scirrhous gastric carcinoma cells. Oncogene (2011) 30, 1413-1421; doi:10.1038/onc.2010.522; published online 15 November 2010

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Genetic and epigenetic alterations during renal carcinogenesis

Arai, Eri, Kanai, Yae

INTERNATIONAL JOURNAL OF CLINICAL AND EXPERIMENTAL PATHOLOGY (E-CENTURY PUBLISHING CORP) 4 ( 1 ) 58 - 73 2011

Research paper (scientific journal), ISSN 1936-2625

Renal cell carcinoma (RCC) is not a single entity, but comprises a group of tumors including clear cell RCC, papillary RCC and chromophobe RCC, which arise from the epithelium of renal tubules. The majority of clear cell RCCs, the major histological subtype, have genetic or epigenetic inactivation of the von Hippel-Lindau (VHL) gene. Germline mutations in the MET and fumarate hydratase (FH) genes lead to the development of type 1 and type 2 papillary RCCs, respectively, and such mutations of either the TSC1 or TSC2 gene increase the risk of RCC. Genome-wide copy number alteration analysis has suggested that loss of chromosome 3p and gain of chromosomes 5q and 7 may be copy number aberrations indispensable for the development of clear cell RCC. When chromosome 1p, 4, 9, 13q or 14q is also lost, more clinicopathologically aggressive clear cell RCC may develop. Since renal carcinogenesis is associated with neither chronic inflammation nor persistent viral infection, and hardly any histological change is evident in corresponding non-tumorous renal tissue from patients with renal tumors, precancerous conditions in the kidney have been rarely described. However, regional DNA hypermethylation on C-type CpG islands has already accumulated in such non-cancerous renal tissues, suggesting that, from the viewpoint of altered DNA methylation, the presence of precancerous conditions can be recognized even in the kidney. Genome-wide DNA methylation profiles in precancerous conditions are basically inherited by the corresponding clear cell RCCs developing in individual patients: DNA methylation alterations at the precancerous stage may further predispose renal tissue to epigenetic and genetic alterations, generate more malignant cancers, and even determine patient outcome. The list of tumor-related genes silenced by DNA hypermethylation has recently been increasing. Genetic and epigenetic profiling provides an optimal means of prognostication for patients with RCCs. Recently developed high-throughput technologies for genetic and epigenetic analyses will further accelerate the identification of key molecules for use in the prevention, diagnosis and therapy of RCCs.

Diagnosis and Prognostication of Ductal Adenocarcinomas of the Pancreas Based on Genome-Wide DNA Methylation Profiling by Bacterial Artificial Chromosome Array-Based Methylated CpG Island Amplification

Gotoh, Masahiro, Arai, Eri, Wakai-Ushijima, Saori, Hiraoka, Nobuyoshi, Kosuge, Tomoo, Hosoda, Fumie, Shibata, Tatsuhiro, Kondo, Tadashi, Yokoi, Sana, Imoto, Issei, Inazawa, Johji, Kanai, Yae

JOURNAL OF BIOMEDICINE AND BIOTECHNOLOGY (HINDAWI PUBLISHING CORPORATION) 2011 780836 2011

Research paper (scientific journal), ISSN 1110-7243

To establish diagnostic criteria for ductal adenocarcinomas of the pancreas (PCs), bacterial artificial chromosome (BAC) array-based methylated CpG island amplification was performed using 139 tissue samples. Twelve BAC clones, for which DNA methylation status was able to discriminate cancerous tissue (T) from noncancerous pancreatic tissue in the learning cohort with a specificity of 100%, were identified. Using criteria that combined the 12 BAC clones, T-samples were diagnosed as cancers with 100% sensitivity and specificity in both the learning and validation cohorts. DNA methylation status on 11 of the BAC clones, which was able to discriminate patients showing early relapse from those with no relapse in the learning cohort with 100% specificity, was correlated with the recurrence-free and overall survival rates in the validation cohort and was an independent prognostic factor by multivariate analysis. Genome-wide DNA methylation profiling may provide optimal diagnostic markers and prognostic indicators for patients with PCs.

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Serous cystic neoplasm in an intrapancreatic accessory spleen (vol 60, 681, 2010)

Hori, Shutaro, Nara, Satoshi, Shimada, Kazuaki, Ojima, Hidenori, Kanai, Yae, Hiraoka, Nobuyoshi

PATHOLOGY INTERNATIONAL (WILEY-BLACKWELL PUBLISHING, INC) 60 ( 12 ) 798 - 798 2010.12

Research paper (scientific journal), ISSN 1320-5463

Association of CYP19A1 polymorphisms with risks for atypical adenomatous hyperplasia and bronchioloalveolar carcinoma in the lungs

Kohno, Takashi, Kakinuma, Ryutaro, Iwasaki, Motoki, Yamaji, Taiki, Kunitoh, Hideo, Suzuki, Kenji, Shimada, Yoko, Shiraishi, Kouya, Kasuga, Yoshio, Hamada, Gerson Shigeaki, Furuta, Koh, Tsuta, Koji, Sakamoto, Hiromi, Kuchiba, Aya, Yamamoto, Seiichiro, Kanai, Yae, Tsugane, Shoichiro, Yokota, Jun

CARCINOGENESIS (OXFORD UNIV PRESS) 31 ( 10 ) 1794 - 1799 2010.10

Research paper (scientific journal), ISSN 0143-3334

Estrogen has been indicated to play an etiological role in the development of lung adenocarcinoma (ADC), particularly bronchioloalveolar carcinoma (BAC), a type of ADC that develops from a benign adenomatous lesion, atypical adenomatous hyperplasia (AAH). Polymorphisms in the CYP19A1 gene cause interindividual differences in estrogen levels. Here, 13 CYP19A1 single-nucleotide polymorphisms (SNPs) were examined for associations with lung AAH risk. AAH is detected as ground-glass opacity (GGO) by computed tomography (CT) examination, and this study consisted of 100 individuals diagnosed with GGO in their lungs among 3088 CT-based cancer screening examinees and 424 without. Minor allele carriers for the rs3764221 SNP showed an elevated risk for GGO [odds ratio (OR) = 1.72, P = 0.017]. Associations of this SNP with risks for lung AAH and BAC in the lungs were next examined using 359 ADC cases whose resected lung lobes were subjected to a histological examination for AAH accompaniment and the presence of BAC components and 330 controls without cancer. The ORs were also increased for lung ADC accompanied by AAH (OR = 1.74, P = 0.029) as well as lung ADC with BAC components (OR = 1.41, P = 0.091). The minor allele was associated with an increased circulating estradiol level (P = 0.079) in a population of 363 postmenopausal women without cancer. These results indicate that CYP19A1 polymorphisms are involved in the risk for lung AAH and BAC in the lungs by causing differences in estrogen levels.

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Serous cystic neoplasm in an intrapancreatic accessory spleen

Shutaro Hori, Satoshi Nara, Kazuaki Shimada, Hidenori Ojima, Yae Kanai, Nobuyoshi Hiraoka

PATHOLOGY INTERNATIONAL (WILEY-BLACKWELL) 60 ( 10 ) 681 - 684 2010.10

Accepted, ISSN 13205463

Serous cystic neoplasm (SCN) of the pancreas is a benign epithelial neoplasm, except in extremely rare malignant cases. Development of SCN in tissues other than the pancreas has been never reported. Here we present the first reported case of SCN in an intrapancreatic accessory spleen (IPAS). A 54-year-old female patient with von Hippel-Lindau (VHL) syndrome was found to have pancreatic tail mass. Pathologically the 25-mm solid mass was an IPAS showing proliferation of clear cuboidal tumor cells without atypia, forming numerous small cysts. The tumor cells were rich in cytoplasmic glycogen and distributed in the splenic tissue almost diffusely. Immunohistochemically, tumor cells were positive for cytokeratins, MUC6, and neuron-specific enolase, and negative for neuroendocrine markers. From these findings, we diagnosed the lesion as SCN in IPAS. This tumor is suggested to develop as a VHL-associated SCN from coexisting pancreatic tissue in IPAS rather than as a metastatic tumor.

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Solitary hepatic lymphangioma: Report of a case

Matsumoto, Takuya, Ojima, Hidenori, Akishima-Fukasawa, Yuri, Hiraoka, Nobuyoshi, Onaya, Hiroaki, Shimada, Kazuaki, Mizuguchi, Yasunori, Sakurai, Shintaro, Ishii, Toshiharu, Kosuge, Tomoo, Kanai, Yae

SURGERY TODAY (SPRINGER) 40 ( 9 ) 883 - 889 2010.09

Research paper (scientific journal), ISSN 0941-1291

A 52-year-old woman presented with upper abdominal pain. Abdominal ultrasonography showed a 4-cm well-defined mass containing solid and cystic components in segment IV of the liver, and contrast-enhanced T1-weighted magnetic resonance imaging revealed heterogeneous enhancement within the tumor, indicating a solid or fibrous component. There were no cystic lesions in any other organs. A partial hepatectomy was performed, based on a preoperative diagnosis of sclerosing hemangioma and biliary cystadenoma or cystadenocarcinoma. Pathologically, the tumor appeared to be a multilocular and cystic lesion lined by attenuated endothelial- like cells with no atypia. Immunohistochemistry demonstrated the endothelial-like cells to be positive for the lymphatic-specific markers D2-40, LYVE-1, and Prox-1, which proved helpful for confirming the diagnosis as solitary hepatic lymphangioma. This case is presented with details of the pathologic and radiologic findings, because solitary hepatic lymphangioma is an extremely rare tumor and no previous reports have provided details of the immunohistochemical characteristics.

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Mice-lacking LMP2, immuno-proteasome subunit, as an animal model of spontaneous uterine leiomyosarcoma

Hayashi, Takuma, Horiuchi, Akiko, Sano, Kenji, Hiraoka, Nobuyoshi, Kanai, Yae, Shiozawa, Tanri, Tonegawa, Susumu, Konishi, Ikuo

PROTEIN & CELL (HIGHER EDUCATION PRESS) 1 ( 8 ) 711 - 717 2010.08

Research paper (scientific journal), ISSN 1674-800X

Uterine tumors are the most common type of gynecologic neoplasm. Uterine leiomyosarcoma (LMS) is rare, accounting for 2% to 5% of tumors of the uterine body. Uterine LMS develops more often in the muscle tissue layer of the uterine body than in the uterine cervix. The development of gynecologic tumors is often correlated with female hormone secretion; however, the development of uterine LMS is not substantially correlated with hormonal conditions, and the risk factors are not yet known. Radiographic evaluation combined with PET/CT can be useless in the diagnosis and surveillance of uterine LMS. Importantly, a diagnostic biomarker, which distinguishes malignant LMS and benign tumor leiomyoma (LMA) is yet to be established. Accordingly, it is necessary to analyze risk factors associated with uterine LMS in order to establish a method of treatment. LMP2-deficient mice spontaneously develop uterine LMS, with a disease prevalence of similar to 40% by 14 months of age. It is therefore of interest whether human uterine LMS shows a loss of LMP2 expression. We found LMP2 expression is absent in human LMS, but present in human LMA. Therefore, defective LMP2 expression may be one of the risk factors for LMS. LMP2 is potentially a diagnostic biomarker for uterine LMS, and gene therapy with LMP2-encording DNA may be a new therapeutic approach.

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Significance of PGP9.5 Expression in Cancer-Associated Fibroblasts for Prognosis of Colorectal Carcinoma

Akishima-Fukasawa, Yuri, Ino, Yoshinori, Nakanishi, Yukihiro, Miura, Ayaka, Moriya, Yoshihiro, Kondo, Tadashi, Kanai, Yae, Hirohashi, Setsuo

AMERICAN JOURNAL OF CLINICAL PATHOLOGY (AMER SOC CLINICAL PATHOLOGY) 134 ( 1 ) 71 - 79 2010.07

Research paper (scientific journal), ISSN 0002-9173

To assess the expression of a cancer-associated fibroblasts (CAFs) marker as an indicator of prognosis, we raised anti protein gene product 9.5 (PGP9.5) monoclonal antibody against cultured fibroblasts. PGP9.5 expression in cultured normal fibroblasts was increased by transforming growth factor 13 stimulation, indicating the phenotypic alteration to activated fibroblast. We immunohistochemically evaluated PGP9.5 expression with the CAFs of 110 colorectal cancer cases under T3 stage. PGP9.5 immunoreactivity in 30% or more of CAFs was defined as high PGP9.5 expression, and the other cases were considered as having low PGP9.5 expression.
Patients with high PGP9.5 expression (42.7%) had significantly shorter survival and a higher incidence of recurrence than the low PGP9.5 expression group (P=.002 and P < .001, respectively). Multivariate analysis indicated PGP9.5 expression as an independent prognostic factor for overall and recurrence-free survival partly as well as lymph node metastasis. These results indicate that PGP9.5 expression in CAFs is a helpful finding to represent the overall biologic behavior of advanced colorectal cancer.

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Metaplastic carcinoma of the breast

Okada, Nao, Hasebe, Takahiro, Iwasaki, Motoki, Tamura, Nobuko, Akashi-Tanaka, Sadako, Hojo, Takashi, Shibata, Tatsuhiro, Sasajima, Yuko, Kanai, Yae, Kinoshita, Takayuki

HUMAN PATHOLOGY (W B SAUNDERS CO-ELSEVIER INC) 41 ( 7 ) 960 - 970 2010.07

Research paper (scientific journal), ISSN 0046-8177

The purposes of this study were to investigate whether the biological characteristics or outcomes of patients with metaplastic carcinoma, invasive ductal carcinoma, or invasive lobular carcinoma of the breast differ; to determine whether the metaplastic carcinoma subtypes have similar malignant potentials; and to identify accurate predictors of outcome in patients with metaplastic carcinoma. The subject comprised 6137 invasive ductal carcinoma patients, 301 invasive lobular carcinoma patients, and 46 metaplastic carcinoma patients of the breast. The metaplastic carcinomas were classified according to the World Health Organization classification. Multivariate analyses clearly demonstrated that the metaplastic carcinoma patients had a significantly poorer outcome than the invasive ductal carcinoma patients or the invasive lobular carcinoma patients independent of the nodal status or age not exceeding 39 years, whereas patients with triple-negative metaplastic carcinomas or triple-negative invasive lobular carcinomas had a poorer outcome than those with triple-negative invasive ductal carcinomas. Although no significant differences in clinical outcome were observed among the metaplastic carcinoma subtypes in multivariate analyses, an age not exceeding 39 years, the presence of skin invasion, and the presence of a squamous cell carcinoma component in nodal tumors were significant outcome predictors for metaplastic carcinoma patients. In conclusion, the results of this study clearly demonstrated that metaplastic carcinoma is more aggressive than invasive ductal carcinoma or invasive lobular carcinoma. Although the metaplastic carcinoma subtypes had no prognostic significance, an age not exceeding 39 years, the presence of skin invasion, and the presence of a squamous cell carcinoma component in nodal tumors were significant predictors of outcome among metaplastic carcinoma patients. (C) 2010 Elsevier Inc. All rights reserved.

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Pancreatic Ducts as an Important Route of Tumor Extension for Acinar Cell Carcinoma of the Pancreas

Ban, Daisuke, Shimada, Kazuaki, Sekine, Shigeki, Sakamoto, Yoshihiro, Kosuge, Tomoo, Kanai, Yae, Hiraoka, Nobuyoshi

AMERICAN JOURNAL OF SURGICAL PATHOLOGY (LIPPINCOTT WILLIAMS & WILKINS) 34 ( 7 ) 1025 - 1036 2010.07

Research paper (scientific journal), ISSN 0147-5185

Acinar cell carcinoma (ACC) of the pancreas is very rare, which usually grows expansively. Recently, a variant of ACC with predominant growth in the pancreatic ducts has been proposed, and is speculated to have potentially less aggressive behavior. The aim of this study was to investigate how the pancreatic duct system is related to the growth and extension of ACC. We reviewed the detailed gross and histologic features of 13 cases of ACC, of which 7 (54%) showed intraductal polypoid growth (IPG) of the tumor in the large pancreatic ducts with a mean IPG length of 24.8 mm. Tumors with IPG were found to spread characteristically along the pancreatic ducts as extending polypoid projections, filling the ducts and destroying the duct walls, although tumors did not tend to extend beyond the pancreatic parenchyma. Comparison of the clinicopathologic characteristics showed that ACC with IPG had less infiltrative features including lymphatic, venous, and neural invasion, formation of tumor thrombus in the portal vein, nodal metastasis, and invasion beyond the pancreas to the surrounding organs; death in only 1 case (14%) of ACC with IPG was the result of ACC itself. In contrast, ACC without IPG frequently showed more infiltrative growth, and was the cause of death in 50% of patients with this type of tumor. Intraductal dissemination of ACC in pancreatic ducts was proven in 1 case of ACC with IPG. These findings suggest that a significant proportion of ACC shows IPG, which is potentially linked to less aggressive clinicopathologic characteristics.

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DNA methylation profiles in precancerous tissue and cancers: carcinogenetic risk estimation and prognostication based on DNA methylation status

Arai, Eri, Kanai, Yae

EPIGENOMICS (FUTURE MEDICINE LTD) 2 ( 3 ) 467 - 481 2010.06

Research paper (scientific journal), ISSN 1750-1911

Alterations in DNA methylation, which are associated with DNA methyltransferase abnormalities and result in silencing of tumor-related genes and chromosomal instability, are involved even in precancerous changes in various organs. DNA methylation alterations also account for the histological heterogeneity and clinicopathological diversity of human cancers. Therefore, we have analyzed DNA methylation on a genome-wide scale in clinical tissue samples. Our approach using the bacterial artificial chromosome array-based methylated CpG island amplification method has revealed that DNA methylation alterations correlated with the future development of more malignant cancers are already accumulated at the precancerous stage in the kidney, liver and urinary tract. DNA methylation profiles at precancerous stages are basically inherited by the corresponding cancers developing in individual patients. Such DNA methylation alterations may confer vulnerability to further genetic and epigenetic alterations, generate more malignant cancers, and thus determine patient outcome. On the basis of bacterial artificial chromosome array-based methylated CpG island amplification data, indicators for carcinogenetic risk estimation have been established using liver tissue specimens from patients with hepatitis virus infection, chronic hepatitis and liver cirrhosis or histologically normal urothelia, and for prognostication using biopsy or surgically resected specimens from patients with renal cell carcinoma, hepatocellular carcinoma and urothelial carcinoma. Such genome-wide DNA methylation profiling has now firmly established the clinical relevance of translational epigenetics.

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CUB Domain-Containing Protein 1, a Prognostic Factor for Human Pancreatic Cancers, Promotes Cell Migration and Extracellular Matrix Degradation

Miyazawa, Yuri, Uekita, Takamasa, Hiraoka, Nobuyoshi, Fujii, Satoko, Kosuge, Tomoo, Kanai, Yae, Nojima, Yoshihisa, Sakai, Ryuichi

CANCER RESEARCH (AMER ASSOC CANCER RESEARCH) 70 ( 12 ) 5136 - 5146 2010.06

Research paper (scientific journal), ISSN 0008-5472

CUB domain-containing protein 1 (CDCP1) is a membrane protein that is highly expressed in several solid cancers. We reported previously that CDCP1 regulates anoikis resistance as well as cancer cell migration and invasion, although the underlying mechanisms have not been elucidated. In this study, we found that expression of CDCP1 in pancreatic cancer tissue was significantly correlated with overall survival and that CDCP1 expression in pancreatic cancer cell lines was relatively high among solid tumor cell lines. Reduction of CDCP1 expression in these cells suppressed extracellular matrix (ECM) degradation by inhibiting matrix metalloproteinase-9 secretion. Using the Y734F mutant of CDCP1, which lacks the tyrosine phosphorylation site, we showed that CDCP1 regulates cell migration, invasion, and ECM degradation in a tyrosine phosphorylation-dependent manner and that these CDCP1-associated characteristics were inhibited by blocking the association of CDCP1 and protein kinase C delta (PKC delta). CDCP1 modulates the enzymatic activity of PKC delta through the tyrosine phosphorylation of PKC delta by recruiting PKC delta to Src family kinases. Cortactin, which was detected as a CDCP1-dependent binding partner of PKC delta, played a significant role in migration and invasion but not in ECM degradation of pancreatic cells. These results suggest that CDCP1 expression might play a crucial role in poor outcome of pancreatic cancer through promotion of invasion and metastasis and that molecules blocking the expression, phosphorylation, or the PKC delta-binding site of CDCP1 are potential therapeutic candidates. Cancer Res; 70(12); 5136-46. (C) 2010 AACR.

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Combined Functional Genome Survey of Therapeutic Targets for Hepatocellular Carcinoma

Satow, Reiko, Shitashige, Miki, Kanai, Yae, Takeshita, Fumitaka, Ojima, Hidenori, Jigami, Takafumi, Honda, Kazufumi, Kosuge, Tomoo, Ochiya, Takahiro, Hirohashi, Setsuo, Yamada, Tesshi

CLINICAL CANCER RESEARCH (AMER ASSOC CANCER RESEARCH) 16 ( 9 ) 2518 - 2528 2010.05

Research paper (scientific journal), ISSN 1078-0432

Purpose: The outcome of patients with advanced hepatocellular carcinoma (HCC) has remained unsatisfactory. Patients with HCC suffer from chronic hepatitis or liver cirrhosis, and their reserve liver function is often limited.
Experimental Design: To develop new therapeutic agents that act specifically on HCC but interfere only minimally with residual liver function, we searched for genes that were upregulated in 20 cases of HCC [namely, discovery sets 1 (n = 10) and 2 (n = 10)] in comparison with corresponding nontumorous liver and a panel representing normal organs using high-density microarrays capable of detecting all exons in the human genome.
Results: Eleven transcripts whose expression was significantly increased in HCC were subjected to siRNA-based secondary screening of genes required for HCC cell proliferation as well as quantitative reverse transcription-PCR analysis [validation sets 1 (n = 20) and 2 (n = 44)] and immunohistochemistry (n = 19). We finally extracted four genes, AKR1B10, HCAP-G, RRM2, and TPX2, as candidate therapeutic targets for HCC. siRNA-mediated knockdown of these candidate genes inhibited the proliferation of HCC cells and the growth of HCC xenografts transplanted into immunodeficient mice.
Conclusions: The four genes we identified were highly expressed in HCC, and HCC cells are highly dependent on these genes for proliferation. Although many important genes must have been overlooked, the selected genes were biologically relevant. The combination of genome-wide expression and functional screening described here is a rapid and comprehensive approach that could be applied in the identification of therapeutic targets in any type of human malignancy. Clin Cancer Res; 16(9); 2518-28. (C) 2010 AACR.

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Establishment of six new human biliary tract carcinoma cell lines and identification of MAGEH1 as a candidate biomarker for predicting the efficacy of gemcitabine treatment

Ojima, Hidenori, Yoshikawa, Daitaro, Ino, Yoshihiro, Shimizu, Hiroko, Miyamoto, Masashi, Kokubu, Akiko, Hiraoka, Nobuyoshi, Morofuji, Noriaki, Kondo, Tadashi, Onaya, Hiroaki, Okusaka, Takuji, Shimada, Kazuaki, Sakamoto, Yoshihiro, Esaki, Minoru, Nara, Satoshi, Kosuge, Tomoo, Hirohashi, Setsuo, Kanai, Yae, Shibata, Tatsuhiro

CANCER SCIENCE (WILEY-BLACKWELL PUBLISHING, INC) 101 ( 4 ) 882 - 888 2010.04

Research paper (scientific journal), ISSN 1347-9032

The aim of this study was to establish new biliary tract carcinoma (BTC) cell lines and identify predictive biomarkers for the potential effectiveness of gemcitabine therapy. Surgical specimens of BTC were transplanted directly into immunodeficient mice to establish xenografts, then subjected to in vitro cell culture. The gemcitabine sensitivity of each cell line was determined and compared with the genome-wide gene expression profile. A new predictive biomarker candidate was validated using an additional cohort of gemcitabine-treated BTC cases. From 55 BTC cases, we established 19 xenografts and six new cell lines. Based on their gemcitabine sensitivity, 10 BTC cell lines (including six new and four publicly available ones) were clearly categorized into two groups, and MAGEH1 mRNA expression in the tumor cells showed a significant negative correlation with their sensitivity to gemcitabine. Immunohistochemically, MAGEH1 protein was detected in three (50%) out of six sensitive cell lines, and four (100%) out of four resistant cell lines. In the validation cohort of gemcitabine-treated recurrence cases, patients were categorized into "effective'' and "non-effective'' groups according to the RECIST guidelines for assessment of chemotherapeutic effects. MAGEH1 protein expression was detected in two (40%) out of five "effective'' cases and all four (100%) "non-effective'' cases. We have established a new BTC bioresource that covers a wide range of biological features, including drug sensitivity, and is linked with clinical information. Negative expression of MAGEH1 protein serves as a potential predictive marker for the effectiveness of gemcitabine therapy in BTC. (Cancer Sci 2010; 101: 882-888)

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Intraductal carcinosarcoma with a heterologous mesenchymal component originating in intraductal papillary-mucinous carcinoma (IPMC) of the pancreas with both carcinoma and osteosarcoma cells arising from IPMC cells

Okamura, Jun, Sekine, Shigeki, Nara, Satoshi, Ojima, Hidenori, Shimada, Kazuaki, Kanai, Yae, Hiraoka, Nobuyoshi

JOURNAL OF CLINICAL PATHOLOGY (B M J PUBLISHING GROUP) 63 ( 3 ) 266 - 269 2010.03

Research paper (scientific journal), ISSN 0021-9746

Carcinosarcoma of the pancreas is extremely rare and its histogenesis is still unclear. This is a report on a 64-year-old female patient with an intraductal carcinosarcoma arising from intraductal papillary-mucinous carcinoma (IPMC) in the pancreas tail. The carcinosarcoma grew as a polypoid mass within the main pancreatic duct. Histologically, the tumour consisted of adenocarcinoma covering the luminal surface of the lesion with minimal stromal invasion, and osteosarcoma occupying the stroma. Immunohistochemical and gene mutation analyses revealed that both the carcinomatous and sarcomatous tumour cells of the carcinosarcoma, as well as the IPMC cells, expressed TP53 and had identical mutations in KRAS and TP53 genes, indicating that these two neoplastic components of the carcinosarcoma shared a common tumorigenesis and arose from the IPMC. This is the first report of a carcinosarcoma originating in IPMC. These findings imply that carcinosarcoma with a heterologous mesenchymal component is of ductal origin.

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Long-term Recurrence-free Survival in a Patient with Primary Hepatic Carcinosarcoma: Case Report with a Literature Review

Yamamoto, Yusuke, Ojima, Hidenori, Shimada, Kazuaki, Onaya, Hiroaki, Hiraoka, Nobuyoshi, Mizuguchi, Yasunori, Kosuge, Tomoo, Kanai, Yae

JAPANESE JOURNAL OF CLINICAL ONCOLOGY (OXFORD UNIV PRESS) 40 ( 2 ) 166 - 173 2010.02

Research paper (scientific journal), ISSN 0368-2811

A 72-year-old man was found to have a 40 mm mass in liver segment VIII during follow-up abdominal ultrasonography for type C viral hepatitis. Abdominal ultrasound showed a well-defined mass containing a cystic component, and computed tomographic hepatic arteriography showed heterogeneous enhancement except for cystic necrosis. Under a pre-operative diagnosis of atypical hepatocellular carcinoma (HCC), partial resection of liver segment VIII was performed. The encapsulated tumor consisted of a peripheral solid component with a central necrotic area. Histologically, the solid component had a two-layer structure, an HCC component in the external area and a sarcomatous component with neoplastic osteoid formation in the internal area, showing histological transition. Immunohistochemically, the HCC component was positive for hepatocyte antigen and negative for vimentin. The Ki-67 labeling index was found to increase from 5% to 58% with increasing histologic atypia. The sarcomatous component was positive for vimentin and negative for pan-keratin and hepatocyte antigen, with a Ki-67 labeling index of > 90%. These findings led to a diagnosis of primary hepatic carcinosarcoma. Although previously reported patients with hepatic carcinosarcoma showed early metastasis with a very poor outcome, this patient has remained free of recurrence for 30 months, which is the longest recurrence-free survival time recorded for this type of cancer. Since relatively early-stage hepatic carcinosarcoma rarely seems to present as a small tumor showing a concentric growth pattern, we report this case with a review of the literature.

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前立腺がん神経温存手術の適応選択に3T MRI拡散強調画像を用いた術前局在診断はどの程度有用か?

原智彦, 三宅基隆, 中西弘之, 河原貴史, 熊谷伸, 堀淳一, 新井恵吏, 金井弥栄, 鶴崎正勝, 込山元清, 中川徹, 藤元博行

日本泌尿器科学会雑誌 ((一社)日本泌尿器科学会) 101 ( 2 ) 290 - 290 2010.02

ISSN 0021-5287

Genome-wide DNA methylation profiles in urothelial carcinomas and urothelia at the precancerous stage

Nishiyama, Naotaka, Arai, Eri, Chihara, Yoshitomo, Fujimoto, Hiroyuki, Hosoda, Fumie, Shibata, Tatsuhiro, Kondo, Tadashi, Tsukamoto, Taiji, Yokoi, Sana, Imoto, Issei, Inazawa, Johji, Hirohashi, Setsuo, Kanai, Yae

CANCER SCIENCE (WILEY-BLACKWELL PUBLISHING, INC) 101 ( 1 ) 231 - 240 2010.01

Research paper (scientific journal), ISSN 1347-9032

To clarify genome-wide DNA methylation profiles during multistage urothelial carcinogenesis, bacterial artificial chromosome (BAC) array-based methylated CpG island amplification (BAMCA) was performed in 18 normal urothelia obtained from patients without urothelial carcinomas (UCs) (C), 17 noncancerous urothelia obtained from patients with UCs (N), and 40 UCs. DNA hypo- and hypermethylation on multiple BAC clones was observed even in N compared to C. Principal component analysis revealed progressive DNA methylation alterations from C to N, and to UCs. DNA methylation profiles in N obtained from patients with invasive UCs were inherited by the invasive UCs themselves, that is DNA methylation alterations in N were correlated with the development of more malignant UCs. The combination of DNA methylation status on 83 BAC clones selected by Wilcoxon test was able to completely discriminate N from C, and diagnose N as having a high risk of carcinogenesis, with 100% sensitivity and specificity. The combination of DNA methylation status on 20 BAC clones selected by Wilcoxon test was able to completely discriminate patients who suffered from recurrence after surgery from patients who did not. The combination of DNA methylation status for 11 BAC clones selected by Wilcoxon test was able to completely discriminate patients with UCs of the renal pelvis or ureter who suffered from intravesical metachronous UC development from patients who did not. Genome-wide alterations of DNA methylation may participate in urothelial carcinogenesis from the precancerous stage to UC, and DNA methylation profiling may provide optimal indicators for carcinogenetic risk estimation and prognostication. (Cancer Sci 2009).

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Fibroblast growth factor receptor 3 mutation in voided urine is a useful diagnostic marker and significant indicator of tumor recurrence in non-muscle invasive bladder cancer

Miyake, Makito, Sugano, Kokichi, Sugino, Hitomi, Imai, Kazuho, Matsumoto, Eri, Maeda, Koshi, Fukuzono, Shinich, Ichikawa, Hiroki, Kawashima, Kiyotaka, Hirabayashi, Kaoru, Kodama, Tetsuro, Fujimoto, Hiroyuki, Kakizoe, Tadao, Kanai, Yae, Fujimoto, Kiyohide, Hirao, Yoshihiko

CANCER SCIENCE (WILEY-BLACKWELL PUBLISHING, INC) 101 ( 1 ) 250 - 258 2010.01

Research paper (scientific journal), ISSN 1347-9032

The fibroblast growth factor receptor (FGFR)-3 gene encodes a receptor tyrosine kinase that is frequently mutated in non-muscle invasive bladder cancer (NMIBC). A sensitive and quantitative assay using peptide nucleic acid-mediated real-time PCR was developed for detecting FGFR3 mutations in the urine samples and evaluated as a molecular marker for detecting intravesical recurrence of NMIBC in patients undergoing transurethral resection of bladder tumor. FGFR3 mutation was examined in tumor tissues and serially taken pre- and postoperative urine sediments in 45 NMIBC patients with a median follow up of 32 months. FGFR3 mutations were detected in 53.3% (24/45) of primary tumor tissues, among which intravesical recurrence developed in 37.5% (9/24) of cases. FGFR3 mutation in the primary tumor was not a significant prognostic indicator for recurrence, while the proportion of FGFR3 mutation (i.e. tumor cellularity was >= 11%) in the preoperative urine sediments was a significant indicator for recurrence in patients with FGFR3 mutations in the primary tumors. FGFR3 mutations were detected in 78% (7/9) of postoperative urine samples from recurrent cases with FGFR3 mutations in the tumor, while no mutations were detected in the urine of 15 non-recurrent cases. Urine cytology was negative in all cases with FGFR3 mutations in the primary tumors, while the sensitivity of cytological examination was as high as 56% (5/9) in cases showing wild-type FGFR3 in the primary tumors. Urine FGFR3 mutation assay and cytological examination may be available in the future as complementary diagnostic modalities in postoperative management of NMIBC. (Cancer Sci 2009).

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Genome-wide DNA methylation profiles in precancerous conditions and cancers.

Kanai Y.

Cancer Sci (WILEY-BLACKWELL PUBLISHING, INC) 101 ( 1 ) 36 - 45 2010

Accepted, ISSN 13479032

Alterations of DNA methylation, which result in chromosomal instability and silencing of tumor-related genes, are among the most consistent epigenetic changes observed in human cancers. Analysis of tissue specimens has revealed that DNA methylation alterations participate in multistage carcinogenesis, even from the early and precancerous stages, especially in association with chronic inflammation and/or persistent viral infection, such as chronic hepatitis or liver cirrhosis resulting from infection with hepatitis B or C virus. DNA methylation alterations can account for the histological heterogeneity and clinicopathological diversity of human cancers. Overexpression of DNA methyltransferase 1 is not a secondary result of increased cell proliferative activity, but is significantly correlated with accumulation of DNA hypermethylation in CpG islands of tumor-related genes. Alteration of DNA methyltransferase 3b splicing may result in chromosomal instability through DNA hypomethylation in pericentromeric satellite regions. Genome-wide analysis of DNA methylation status has revealed that the DNA methylation profile at the precancerous stage is basically inherited by the corresponding cancers developing in individual patients. DNA methylation status is not simply altered at the precancerous stage; rather, DNA methylation alterations at the precancerous stage may confer vulnerability to further genetic and epigenetic alterations, generate more malignant cancers, and thus determine patient outcome. Therefore, genome-wide DNA methylation profiling may provide optimal indicators for carcinogenetic risk estimation and prognostication, and thus provide an avenue for cancer prevention and therapy on an individual basis. (Cancer Sci 2009).

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Tumour necrosis is a postoperative prognostic marker for pancreatic cancer patients with a high interobserver reproducibility in histological evaluation.

Hiraoka N, Ino Y, Sekine S, Tsuda H, Shimada K, Kosuge T, Zavada J, Yoshida M, Yamada K, Koyama T, Kanai Y.

Br J Cancer (NATURE PUBLISHING GROUP) 103 ( 7 ) 1057 - 1065 2010

Accepted, ISSN 00070920

BACKGROUND: Tumour necrosis reflects the presence of hypoxia, which can be indicative of an aggressive tumour phenotype. The aim of this study was to investigate whether histological necrosis is a useful predictor of outcome in patients with pancreatic ductal carcinoma (PDC).
METHODS: We reviewed histopathological findings in 348 cases of PDC in comparison with clinicopathological information. We counted small necrotic foci (micronecrosis) as necrosis, in addition to massive necrosis that had been only defined as necrosis in previous studies. The reproducibility of identifying histological parameters was tested by asking five independent observers to blindly review 51 examples of PDC.
RESULTS: Both micronecrosis and massive necrosis corresponded to hypoxic foci expressing carbonic anhydrase IX detected by immunohistochemistry. Multivariate survival analysis showed that histological necrosis was an independent predictor of poor outcome in terms of both disease-free survival (DFS) and disease-specific survival (DSS) of PDC patients. In addition, metastatic status, and lymphatic, venous, and intrapancreatic neural invasion were independent prognostic factors for shorter DFS and metastatic status, margin status, lymphatic invasion, and intrapancreatic neural invasion were independent prognostic factors for DSS. The interobserver reproducibility of necrosis identification among the five independent observers was 'almost perfect' (kappa-value of 0.87).
CONCLUSION: Histological necrosis is a simple, accurate, and reproducible predictor of postoperative outcome in PDC patients. British Journal of Cancer (2010) 103, 1057-1065. doi:10.1038/sj.bjc.6605854 www.bjcancer.com Published online 24 August 2010 (C) 2010 Cancer Research UK

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Genome-wide DNA methylation profiles in liver tissue at the precancerous stage and in hepatocellular carcinoma

Arai, Eri, Ushijima, Saori, Gotoh, Masahiro, Ojima, Hidenori, Kosuge, Tomoo, Hosoda, Fumie, Shibata, Tatsuhiro, Kondo, Tadashi, Yokoi, Sana, Imoto, Issei, Inazawa, Johji, Hirohashi, Setsuo, Kanai, Yae

INTERNATIONAL JOURNAL OF CANCER (WILEY-LISS) 125 ( 12 ) 2854 - 2862 2009.12

Research paper (scientific journal), ISSN 0020-7136

To clarify genome-wide DNA methylation profiles during hepatocarcinogenesis, bacterial artificial chromosome (BAC) array-based methylated CpG island amplification was performed on 126 tissue samples. The average numbers of BAC clones showing DNA hypo- or hypermethylation increased from noncancerous liver tissue obtained from patients with hepatocellular carcinomas (HCCs) (N) to HCCs. N appeared to be at the precancerous stage, showing DNA methylation alterations that were correlated with the future development of HCC. Using Wilcoxon test, 25 BAC clones, whose DNA methylation status was inherited by HCCs from N and were able to discriminate 15 N samples from 10 samples of normal liver tissue obtained from patients without HCCs (C) with 100% sensitivity and specificity, were identified. The criteria using the 25 BAC clones were able to discriminate 24 additional N samples from 26 C samples in the validation set with 95.8% sensitivity and 96.2% specificity. Using Wilcoxon test, 41 BAC clones, whose DNA methylation status was able to discriminate patients who survived more than 4 years after hepatectomy from patients who suffered recurrence within 6 months and died within a year after hepatectomy, were identified. The DNA methylation status of the 41 BAC clones was correlated with the cancer-free and overall survival rates of patients with HCC. Multivariate analysis revealed that satisfying the criteria using the 41 BAC clones was an independent predictor of overall outcome. Genome-wide alterations of DNA methylation may participate in hepatocarcinogenesis from the precancerous stage, and DNA methylation profiling may provide optimal indicators for carcinogenetic risk estimation and prognostication. (C) 2009 UICC

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Dicer is required for proper liver zonation

Sekine, Shigeki, Ogawa, Reiko, Mcmanus, Michael T., Kanai, Yae, Hebrok, Matthias

JOURNAL OF PATHOLOGY (JOHN WILEY & SONS LTD) 219 ( 3 ) 365 - 372 2009.11

Research paper (scientific journal), ISSN 0022-3417

A number of genes and their protein products are expressed within the liver lobules in a region-specific manner and confer heterogeneous metabolic properties to hepatocytes; this phenomenon is known as 'metabolic zonation'. To elucidate the roles of Dicer, an endoribonuclease III type enzyme required for microRNA biogenesis, in the establishment of liver zonation, we examined the distribution of proteins exhibiting pericentral or periportal localization in hepatocyte-specific Dicer1 knockout mouse livers. Immunohistochemistry showed that the localization of pericentral proteins was mostly preserved in Dicer1-deficient livers. However, glutamine synthetase, whose expression is normally confined to a few layers of hepatocytes surrounding the central veins, was expressed in broader pericentral areas. Even more striking was the observation that all the periportal proteins that were examined, including phosphoenolpyruvate carboxykinase, E-cadherin, arginase 1, and carbamoyl phosphate synthetase-I, lost their localized expression patterns and were diffusely expressed throughout the entire lobule. Thus, with regard to periportal protein expression, the consequences of Dicer loss were similar to those caused by the disruption of beta-catenin. An analysis of livers deficient in beta-catenin did not identify the down-regulation of Dicer1 or any microRNAs, indicating that they are not directly activated by beta-catenin. Thus, the present study illustrates that Dicer plays a pivotal role in the establishment of liver zonation. Dicer is essential for the suppression of periportal proteins by Wnt/beta-catenin/TCF signalling, albeit it likely acts in an indirect manner. Copyright (C) 2009 Pathological Society of Great Britain and Ireland. Published by lolin Wiley & Sons, Ltd.

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Characteristics of prostate cancers found in specimens removed by radical cystoprostatectomy for bladder cancer and their relationship with serum prostate-specific antigen level

Nakagawa, Tohru, Kanai, Yae, Komiyama, Motokiyo, Fujimoto, Hiroyuki, Kakizoe, Tadao

CANCER SCIENCE (WILEY-BLACKWELL PUBLISHING, INC) 100 ( 10 ) 1880 - 1884 2009.10

Research paper (scientific journal), ISSN 1347-9032

Prostate cancer mass screening using serum prostate-specific antigen (PSA) has been conducted widely in the world. However, little is known about the true prevalence of prostate cancer in the 'normal' PSA range (4.0 ng/mL or less). The aim of the present study was to elucidate the clinicopathological features of prostate cancer occurring in men with a wide range of PSA levels. The study comprised 349 male patients who underwent radical cystoprostatectomy for bladder cancer. Patients who had had treatment for known prostate cancer were excluded. Tissue specimens were reviewed microscopically. Ninety-one patients (26.1%) were found to have prostate cancer, and 68 (74.7%) of these 91 cancers were considered to be clinically significant. Both increasing patient age and PSA level were significantly correlated with an increased incidence of both all and significant prostate cancers. Sixty-five (21.9%) among 297 patients with PSA < 4.0 ng/mL had prostate cancer, and 45 (69.2%) of the 65 cancers were significant cancers. Eighteen patients had prostate cancers 0.5 mL or more in volume. Among the 18 patients, the PSA level was 4 ng/mL or more in 11, and 3 ng/mL or more in 15. Our study shows that prostate cancer is a common finding in radical cystoprostatectomy specimens excised because of bladder cancers, and a significant proportion of these cancers are clinically significant. PSA still appears to be a useful screening tool for detecting prostate cancers with significant volume. (Cancer Sci 2009; 100: 1880-1884).

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Quantitative proteomics using formalin-fixed paraffin-embedded tissues of oral squamous cell carcinoma

Negishi, Ayako, Masuda, Mari, Ono, Masaya, Honda, Kazufumi, Shitashige, Miki, Satow, Reiko, Sakuma, Tomohiro, Kuwabara, Hideya, Nakanishi, Yukihiro, Kanai, Yae, Omura, Ken, Hirohashi, Setsuo, Yamada, Tesshi

CANCER SCIENCE (WILEY-BLACKWELL PUBLISHING, INC) 100 ( 9 ) 1605 - 1611 2009.09

Research paper (scientific journal), ISSN 1347-9032

Clinical proteomics using a large archive of formalin-fixed paraffin-embedded (FFPE) tissue blocks has long been a challenge. Recently, a method for extracting proteins from FFPE tissue in the form of tryptic peptides was developed. Here we report the application of a highly sensitive mass spectrometry (MS)-based quantitative proteome method to a small amount of samples obtained by laser microdissection from FFPE tissues. Cancerous and adjacent normal epithelia were microdissected from FFPE tissue blocks of 10 squamous cell carcinomas of the tongue. Proteins were extracted in the form of tryptic peptides and analyzed by 2-dimensional image-converted analysis of liquid chromatography and mass spectrometry (2DICAL), a label-free quantitative proteomics method developed in our laboratory. From a total of 25 018 peaks we selected 72 mass peaks whose expression differed significantly between cancer and normal tissues (P < 0.001, paired t-test). The expression of transglutaminase 3 (TGM3) was significantly down-regulated in cancer and correlated with loss of histological differentiation. Hypermethylation of TGM3 gene CpG islands was observed in 12 oral squamous cell carcinoma (OSCC) cell lines with reduced TGM3 expression. These results suggest that epigenetic silencing of TGM3 plays certain roles in the process of oral carcinogenesis. The method for quantitative proteomic analysis of FFPE tissue described here offers new opportunities to identify disease-specific biomarkers and therapeutic targets using widely available archival samples with corresponding detailed pathological and clinical records. (Cancer Sci 2009; 100: 1605-1611).

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Prognostic Significance of CXCL12 Expression in Patients With Colorectal Carcinoma

Akishima-Fukasawa, Yuri, Nakanishi, Yukihiro, Ino, Yoshinori, Moriya, Yoshihiro, Kanai, Yae, Hirohashi, Setsuo

AMERICAN JOURNAL OF CLINICAL PATHOLOGY (AMER SOC CLINICAL PATHOLOGY) 132 ( 2 ) 202 - 210 2009.08

Research paper (scientific journal), ISSN 0002-9173

The present study investigated the protein expression level of CXCL12 in colorectal cancer and aimed to elucidate its association with prognosis. CXCL 12 positivity in 50% or more of tumor cells was defined as high expression and that in less than 50% of the tumor cells as low expression. CXCL12+ tumor budding at the invasive from was divided into 2 grades: high with 10 or more budding foci per x 200 field of view and low grade with fewer than 10 budding foci. Patients with high expression (72.7%) and high grade CXCL12+ tumor budding (43.0%) had significantly shorter survival than patients with low expression (P = .014) and low grade (P = .003), respectively. Patients with a combination of high expression and high grade had the worst outcome (P < .001). Our study demonstrated that CXCL12 expression in colorectal cancer cells and at sites of budding were significant prognostic factors. Furthermore, together with lymph node metastasis, a combination of both expression patterns was a more powerful independent prognostic factor.

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Disruption of Dicer1 Induces Dysregulated Fetal Gene Expression and Promotes Hepatocarcinogenesis

Sekine, Shigeki, Ogawa, Reiko, Ito, Rie, Hiraoka, Nobuyoshi, McManus, Michael T., Kanai, Yae, Hebrok, Matthias

GASTROENTEROLOGY (W B SAUNDERS CO-ELSEVIER INC) 136 ( 7 ) 2304 - 2315 2009.06

Research paper (scientific journal), ISSN 0016-5085

Background & Aims: Growing evidence suggests that microRNAs coordinate various biological processes in the liver. We describe experiments to address the physiologic roles of these new regulators of gene expression in the liver that are as of yet largely undefined. Methods: We disrupted Dicer, an enzyme essential for the processing of microRNAs, in hepatocytes using a conditional knockout mouse model to elucidate the consequences of loss of microRNAs. Results: The conditional knockout mouse livers showed the efficient disruption of Dicer1 at 3 weeks after birth. This resulted in prominent steatosis and the depletion of glycogen storage. Dicer1-deficient liver exhibited increased growth-promoting gene expression and the robust expression of fetal stage-specific genes. The consequence of Dicer elimination included both increased hepatocyte proliferation and overwhelming apoptosis. Over time, Dicer1-expressing wild-type hepatocytes that had escaped Cre-mediated recombination progressively repopulated the entire liver. Unexpectedly, however, two thirds of the mutant mice spontaneously developed hepatocellular carcinomas derived from residual Dicer1-deficient hepatocytes at 1 year of age. Conclusions: Dicer and microRNAs have critical roles in hepatocyte survival, metabolism, developmental gene regulation, and tumor suppression in the liver. Loss of Dicer primarily impairs hepatocyte survival but can promote hepatocarcinogenesis in cooperation with additional oncogenic stimuli.

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Esophageal melanomas harbor frequent NRAS mutations unlike melanomas of other mucosal sites

Sekine, Shigeki, Nakanishi, Yukihiro, Ogawa, Reiko, Kouda, Satoko, Kanai, Yae

VIRCHOWS ARCHIV (SPRINGER) 454 ( 5 ) 513 - 517 2009.05

Research paper (scientific journal), ISSN 0945-6317

Mucosal melanomas have genetic alterations distinct from those in cutaneous melanomas. For example, NRAS- and BRAF-activating mutations occur frequently in cutaneous melanomas, but not in mucosal melanomas. We examined 16 esophageal melanomas for genetic alterations in NRAS, BRAF, and KIT to determine whether they exhibit genetic features common to melanomas arising from other mucosal sites. A sequencing analysis identified NRAS mutations in six cases; notably, four of these mutations were located in exon 1, an uncommon mutation site in cutaneous and other mucosal melanomas. BRAF and KIT mutations were found in one case each. Immunohistochemistry showed KIT expression in four cases, including the tumor with a KIT mutation and two other intramucosal tumors. The low frequency of BRAF mutations and the presence of a KIT mutation-positive case are findings similar to those of mucosal melanomas of other sites, but the prevalence of NRAS mutations was even higher than that of cutaneous melanomas. The present study implies that esophageal melanomas have genetic alterations unique from those observed in other mucosal melanomas.

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Genome-wide DNA methylation profiles in both precancerous conditions and clear cell renal cell carcinomas are correlated with malignant potential and patient outcome

Arai, Eri, Ushijima, Saori, Fujimoto, Hiroyuki, Hosoda, Fumie, Shibata, Tatsuhiro, Kondo, Tadashi, Yokoi, Sana, Imoto, Issei, Inazawa, Johji, Hirohashi, Setsuo, Kanai, Yae

CARCINOGENESIS (OXFORD UNIV PRESS) 30 ( 2 ) 214 - 221 2009.02

Research paper (scientific journal), ISSN 0143-3334

To clarify genome-wide DNA methylation profiles during multistage renal carcinogenesis, bacterial artificial chromosome array-based methylated CpG island amplification (BAMCA) was performed. Non-cancerous renal cortex tissue obtained from patients with clear cell renal cell carcinomas (RCCs) (N) was at the precancerous stage where DNA hypomethylation and DNA hypermethylation on multiple bacterial artificial chromosome (BAC) clones were observed. By unsupervised hierarchical clustering analysis based on BAMCA data for their N, 51 patients with clear cell RCCs were clustered into two subclasses, Clusters A(N) (n = 46) and B-N (n = 5). Clinicopathologically aggressive clear cell RCCs were accumulated in Cluster B-N, and the overall survival rate of patients in Cluster B-N was significantly lower than that of patients in Cluster A(N). By unsupervised hierarchical clustering analysis based on BAMCA data for their RCCs, 51 patients were clustered into two subclasses, Clusters A(T) (n = 43) and B-T (n = 8). Clinicopathologically aggressive clear cell RCCs were accumulated in Cluster B-T, and the overall survival rate of patients in Cluster B-T was significantly lower than that of patients in Cluster A(T). Multivariate analysis revealed that belonging to Cluster B-T was an independent predictor of recurrence. Cluster B-N was completely included in Cluster B-T, and the majority of the BAC clones that significantly discriminated Cluster B-N from Cluster A(N) also discriminated Cluster B-T from Cluster A(T). In individual patients, DNA methylation status in N was basically inherited by the corresponding clear cell RCC. DNA methylation alterations in the precancerous stage may generate more malignant clear cell RCCs and determine patient outcome.

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Mucus Rupture (Extrusion) and Duct Expansion/Expansive Growth are not Diagnostic of Minimal Invasion When Seen With Intraductal Papillary Mucinous Neoplasms Reply

Nara, Satoshi, Shimada, Kazuaki, Kosuge, Tomoo, Kanai, Yae, Hiraoka, Nobuyoshi

AMERICAN JOURNAL OF SURGICAL PATHOLOGY (LIPPINCOTT WILLIAMS & WILKINS) 33 ( 2 ) 321 - 322 2009.02

Research paper (scientific journal), ISSN 0147-5185

Intraductal carcinoma component as a favorable prognostic factor in biliary tract carcinoma

Ojima, Hidenori, Kanai, Yae, Iwasaki, Motoki, Hiraoka, Nobuyoshi, Shimada, Kazuaki, Sano, Tsuyoshi, Sakamoto, Yoshihiro, Esaki, Minoru, Kosuge, Tomoo, Sakamoto, Michiie, Hirohashi, Setsuo

CANCER SCIENCE 100 ( 1 ) 62 - 70 2009.01

Research paper (scientific journal), Accepted, ISSN 1347-9032

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Chromatin remodeling at Alu repeats by epigenetic treatment activates silenced microRNA-512-5p with downregulation of Mcl-1 in human gastric cancer cells.

Saito Y, Suzuki H, Tsugawa H, Nakagawa I, Matsuzaki J, Kanai Y, Hibi T.

Oncogene (NATURE PUBLISHING GROUP) 28 ( 30 ) 2738 - 2744 2009

Accepted, ISSN 09509232

Epigenetic therapy using DNA methylation inhibitors and histone deacetylase (HDAC) inhibitors has clinical promise for the treatment of human malignancies. To investigate roles of microRNAs (miRNAs) on epigenetic therapy of gastric cancer, the miRNA expression profile was analysed in human gastric cancer cells treated with 5-aza-2'-deoxycytidine (5-Aza-CdR) and 4-phenylbutyric acid (PBA). miRNA microarray analysis shows that most of miRNAs activated by 5-Aza-CdR and PBA in gastric cancer cells are located at Alu repeats on chromosome 19. Analyses of chromatin modification show that DNA demethylation and HDAC inhibition at Alu repeats activates silenced miR-512-5p by RNA polymerase II. In addition, activation of miR-512-5p by epigenetic treatment induces suppression of Mcl-1, resulting in apoptosis of gastric cancer cells. These results suggest that chromatin remodeling at Alu repeats plays critical roles in the regulation of miRNA expression and that epigenetic activation of silenced Alu-associated miRNAs could be a novel therapeutic approach for gastric cancer. Oncogene (2009) 28, 2738-2744; doi:10.1038/onc.2009.140; published online 8 June 2009

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MicroRNA-500 as a potential diagnostic marker for hepatocellular carcinoma

Yamamoto, Yusuke, Kosaka, Nobuyoshi, Tanaka, Minoru, Koizumi, Fumiaki, Kanai, Yae, Mizutani, Takayuki, Murakami, Yoshiki, Kuroda, Masahiko, Miyajima, Atsushi, Kato, Takashi, Ochiya, Takahiro

BIOMARKERS (TAYLOR & FRANCIS LTD) 14 ( 7 ) 529 - 538 2009

Research paper (scientific journal), ISSN 1354-750X

We identified that microRNA expression changed dynamically during liver development and found that miR-500 is an oncofetal miRNA in liver cancer. miR-500 was abundantly expressed in several human liver cancer cell lines and 45% of human hepatocellular carcinoma (HCC) tissue. Most importantly, an increased amount of miR-500 was found in the sera of the HCC patients. In fact, miR-500 levels in sera of the HCC patients returned to normal after the surgical treatment in three HCC patients. Our findings reveal that diverse changes of miRNAs occur during liver development and, one of these, miR-500 is an oncofetal miRNA relevant to the diagnosis of human HCC.

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Podoplanin Expression Identified in Stromal Fibroblasts as a Favorable Prognostic Marker in Patients with Colorectal Carcinoma

Yamanashi, Takahiro, Nakanishi, Yukihiro, Fujii, Gen, Akishima-Fukasawa, Yuri, Moriya, Yoshihiro, Kanai, Yae, Watanabe, Masahiko, Hirohashi, Setsuo

ONCOLOGY (KARGER) 77 ( 1 ) 53 - 62 2009

Research paper (scientific journal), ISSN 0030-2414

Objective: The microenvironment of cancer plays a critical role in its progression. However, the molecular features of cancer-associated fibroblasts (CAFs) are less well understood than those of cancer cells. We investigated the clinicopathological significance of podoplanin expression in stromal fibroblasts in patients with colorectal cancer (CRC). Methods: We selected podoplanin as an upregulated marker in CAF from a DNA microarray experiment. Consequently, podoplanin was identified as an upregulated gene. Immunohistochemical podoplanin expression was investigated at the National Cancer Center Hospital, Tokyo, Japan, in 120 patients with advanced CRC, and its clinicopathological significance was examined. The biological function of podoplanin expression was also assessed by a coculture invasion assay with CRC cell lines such as HCT116 and HCT15. Results: Podoplanin expression was exclusively confined to stromal fibroblasts and absent in tumor cells. Podoplanin is absent in normal stroma except for lymphatic vessels. Staining was considered positive when over 30% of the cancer stroma was stained. Positive podoplanin expression was significantly correlated with a more distal tumor localization (p = 0.013) and a shallower depth of tumor invasion (p = 0.011). Univariate analysis revealed that negative podoplanin expression in stromal fibroblasts was significantly associated with reduced disease-specific survival (p = 0.0017) and disease-free survival (p < 0.0001). Multivariate analysis revealed that negative podoplanin expression (p = 0.016) and lymph node metastasis (p = 0.027) were significantly associated with disease-free survival. CRC cell invasion was augmented by coculture with CAFs that were treated with siRNA for podoplanin. Conclusions: Our results suggest that a positive podoplanin expression in stromal fibroblasts could have a protective role against CRC cell invasion and is a significant indicator of a good prognosis in patients with advanced CRC, supported by biological analysis showing that podoplanin expression in CAFs is associated with decreased CRC cell invasion. Copyright (C) 2009 S. Karger AG, Basel

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OP-459 2002年TNM分類におけるpT3腎細胞癌の亜分類の再考(腎腫瘍/診断・マーカー,一般演題口演,第97回日本泌尿器科学会総会)

中川徹, 新井恵吏, 熊谷伸, 堀淳一, 根岸孝仁, 中西弘之, 込山元清, 金井弥栄, 藤元博行

日本泌尿器科学会雑誌 (一般社団法人日本泌尿器科学会) 100 ( 2 ) 303 - 303 2009

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Alterations of DNA methylation and clinicopathological diversity of human cancers

Kanai, Yae

PATHOLOGY INTERNATIONAL (WILEY-BLACKWELL) 58 ( 9 ) 544 - 558 2008.09

Research paper (scientific journal), ISSN 1320-5463

Alterations of DNA methylation can account for the histological heterogeneity, reflected in the stepwise progression and complex biological characteristics of human cancers, that genetic alterations alone cannot explain. Analysis of DNA methylation status in tissue samples can be an aid to understanding the molecular mechanisms of multistage carcinogenesis. Human cancer cells show a drastic change in DNA methylation status, that is, overall DNA hypomethylation and regional DNA hypermethylation, which results in chromosomal instability and silencing of tumor-suppressor genes. Overexpression of DNA methyltransferase (DNMT) 1 is not a secondary result of increased cell proliferative activity but may underline the CpG island methylator phenotype of cancers. Splicing alteration of DNMT3B may result in chromosomal instability through DNA hypomethylation of pericentromeric satellite regions. Alterations of DNA methylation are observed even in the precancerous stage frequently associated with chronic inflammation and/or persistent viral infection or with cigarette smoking. Precancerous conditions showing alterations of DNA methylation may generate more malignant cancers. Aberrant DNA methylation is significantly associated with aggressiveness of cancers and poorer outcome of cancer patients. Genome-wide analysis of DNA methylation status based on array-based technology may identify DNA methylation profiles that can be used as appropriate indicators for carcinogenetic risk estimation and prognostication.

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Genetic clustering of clear cell renal cell carcinoma based on array-comparative genomic hybridization: Its association with DNA methylation alteration and patient outcome

Arai, Ed, Ushijima, Saori, Tsuda, Hitoshi, Fujimoto, Hiroyuki, Hosoda, Fumie, Shibata, Tatsuhiro, Kondo, Tadashi, Imoto, Issei, Inazawa, Johji, Hirohashi, Setsuo, Kanai, Yae

CLINICAL CANCER RESEARCH (AMER ASSOC CANCER RESEARCH) 14 ( 17 ) 5531 - 5539 2008.09

Research paper (scientific journal), ISSN 1078-0432

Purpose:The aim of this study was to clarify genetic and epigenetic alterations occurring during renal carcinogenesis.
Experimental Design: Copy number alterations were examined by array-based comparative genomic hybridization analysis using an array harboring 4,361 bacterial artificial chromosome clones, and DNA methylation alterations on CpG islands of the p16 human MutL homologue I von Hippel-Lindau, and thrombospondin 1 genes and the methylated in tumor (MINT-11, MINT-2, MINT-112, MINT-25, and MINT-31) clones were examined in 51 clear cell renal cell carcinomas (RCC).
Results: By unsupervised hierarchical clustering analysis based on copy number alterations, clear cell RCCs were clustered into the two subclasses, clusters A (n = 34) and B (n = 17). Copy number alterations were accumulated in cluster B. Loss of chromosome 3p and gain of 5q and 7 were frequent in both clusters A and B, whereas loss of 1p, 4, 9,13q, and 14q was frequent only in cluster B. The average number of methylated CpG islands in cluster B was significantly higher than those in clusterA. Clear cell RCCs showing higher histologic grades, vascular involvement, renal vein tumor thrombi, and higher pathologic stages were accumulated in cluster B. The recurrence-free and overall survival rates of patients in cluster B were significantly lower than those of patients in cluster A. Multivariate analysis revealed that genetic clustering was a predictor of recurrence-free survival and was independent of histologic grade and pathologic stage.
Conclusions: This genetic clustering of clear cell RCC is significantly associated with regional DNA hypermethylation and may become a prognostic indicator for patients with RCC.

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肝癌バイオマーカー候補因子microRNA-500の同定と解析(Identification of a novel liver cancer biomarker candidate, miR-500, designated as an oncofetal microRNA)

小坂展慶, 山本雄介, 宮島篤, 小泉史明, 水谷隆之, 金井弥栄, 落谷孝広

日本癌学会総会記事 (日本癌学会) 67回 413 - 413 2008.09

Accepted, ISSN 0546-0476

Molecular mechanisms of uterine leiomyosarcomas: involvement of defect in LMP2 expression.

Hayashi T, Shimamura Y, Saegusa T, Horiuchi A, Kobayashi Y, Hiraoka N, Kanai Y, Aburatani H, Sano K, Konishi I

Gene regulation and systems biology (Gene Regulation and Systems Biology) 2008 ( 2 ) 297 - 305 2008.07

Accepted

Patients with uterine leiomyosarcoma (LMS) typically present with vaginal bleeding, pain, and a pelvic mass. Typical presentations with hypercalcemia or eosinophilia have been reported. Radiographic evaluation with combined positron emission tomography/computed tomography may assist in the diagnosis and surveillance of women with uterine LMS. A recently developed risk-assessment index is highly predictive of disease-specific survival. Ovarian preservation does not appear to negatively impact outcome, and the addition of adjuvant therapy after surgical treatment does not seem to improve survival. It is noteworthy that LMP2-deficient mice exhibit spontaneous development of uterine LMS with a disease prevalence of approximately 37% by 12 months of age. The LMP2 gene is transcribed from a promoter containing an interferon (IFN)-gamma-response factor element; thus, the IFN-gamma-signal strongly induces LMP2 expression. Furthermore, a recent report demonstrated the loss of ability to induce LMP2 expression, which is an interferon (IFN)-gamma-inducible factor, in human uterine LMS tissues and cell lines. Analysis of human uterine LMS shows somatic mutations in the IFN gamma signalling pathway, thus the loss of LMP2 induction is attributable to a defect in the earliest steps of the IFN-gamma signalling pathway. The discovery of an impaired key cell-signalling pathway may provide new targets for diagnostic approaches and therapeutic intervention.

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Frequent inactivation of a putative tumor suppressor, angiopoietin-like protein 2, in ovarian cancer

Kikuchi, Ryoko, Tsuda, Hitoshi, Kozaki, Ken-ichi, Kanai, Yae, Kasamatsu, Takahiro, Sengoku, Kazuo, Hirohashi, Setsuo, Inazawa, Johji, Imoto, Issei

CANCER RESEARCH (AMER ASSOC CANCER RESEARCH) 68 ( 13 ) 5067 - 5075 2008.07

Research paper (scientific journal), ISSN 0008-5472

Angiopoietin-like protein 2 (ANGPTL2) is a secreted protein belonging to the angiopoietin family, the members of which are implicated in various biological processes, although its receptor remains unknown. We identified a homozygous loss of ANGPTL2 (9q33.3) in the course of screening a panel of ovarian cancer (OC) cell lines for genomic copy-number aberrations using in-house array-based comparative genomic hybridization. ANGPTL2 mRNA expression was observed in normal ovarian tissue and immortalized normal ovarian epithelial cells, but was reduced in some OC lines without its homozygous deletion (18 of 23 lines) and restored after treatment with 5-aza 2'-deoxycytidine. The methylation status of sequences around the ANGPTL2 CpG-island with clear promoter activity inversely correlated with expression. ANGPTL2 methylation was frequently observed in primary OC tissues as well. In an immunohistochemical analysis of primary OCs, ANGPTL2 expression was frequently reduced (51 of 100 cases), and inversely correlated with methylation status. Patients with OC showing reduced ANGPTL2 immunoreactivity had significantly worse survival in the earlier stages (stages I and II), but better survival in advanced stages (stages III and IV). The restoration of ANGPTL2 expression or treatment with conditioned medium containing ANGPTL2 inhibited the growth of OC cells originally lacking the expression of this gene, whereas the knockdown of endogenous ANGPTL2 accelerated the growth of OC cells with the expression of ANGPTL2. These results suggest that, at least partly, epigenetic silencing by hypermethylation of the ANGPTL2 promoter leads to a loss of ANGPTL2 function, which may be a factor in the carcinogenesis of OC in a stage-dependent manner.

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CUB-domain-containing protein 1 regulates peritoneal dissemination of gastric scirrhous carcinoma

Uekita, Takamasa, Tanaka, Masamitsu, Takigahira, Misato, Miyazawa, Yuri, Nakanishi, Yukihiro, Kanai, Yae, Yanagihara, Kazuyoshi, Sakai, Ryuichi

AMERICAN JOURNAL OF PATHOLOGY (AMER SOC INVESTIGATIVE PATHOLOGY, INC) 172 ( 6 ) 1729 - 1739 2008.06

Research paper (scientific journal), ISSN 0002-9440

CUB-domain-containing protein 1 (CDCP1) is a type-I transmembrane protein that is highly expressed in colon, breast, and lung cancers. We recently revealed that CDCP1 is associated with and phosphorylated by Src family kinases and is involved in the regulation of anchorage independence of certain lung cancer cell lines. in this study, we examined whether CDCP1 is involved in the regulation of tumor progression of scirrhous gastric cancer, which is a diffusely infiltrative carcinoma with high invasion potential. Expression and phosphorylation levels of CDCP1 correlated with the invasive potential of scirrhous; gastric cancers. Reduction of CDCP1 expression by siRNA suppressed migration, invasion, and anchorage independence without affecting the proliferation of highly invasive scirrhous gastric cancer cells. However, CDCP1 overexpression promoted gastric cancer cell migration with low potential of invasion. Loss of CDCP1 suppressed invasion and dissemination of cancer cells that were orthotopically implanted in the gastric wall of nude mice. Expression and phosphorylation of CDCP1 were also detected in cancer cells of surgically resected tissues of human scirrhous gastric cancer by immunohistochemical analysis. Our results suggest that CDCP1 promotes invasion and peritoneal dissemination of cancer cells through the regulation of cell migration and anchorage independence. Therefore, it is both a potential prognostic and therapeutic target in certain types of gastrointestinal cancers, and suppression of its phosphorylation might be a useful strategy for modulating cancer metastasis.

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Overexpression of HDACs: a prognostic marker for gastric cancer identified by tissue microarray

Kanai, Yae

LANCET ONCOLOGY 9 ( 2 ) 91 - 93 2008.02

Research paper (scientific journal), ISSN 1470-2045

Minimally invasive intraductal papillary-mucinous carcinoma of the pancreas: Clinicopathologic study of 104 intraductal papillary-mucinous neoplasms

Nara, Satoshi, Shimada, Kazuaki, Kosuge, Tonioo, Kanai, Yae, Hiraoka, Nobuyoshi

AMERICAN JOURNAL OF SURGICAL PATHOLOGY (LIPPINCOTT WILLIAMS & WILKINS) 32 ( 2 ) 243 - 255 2008.02

Research paper (scientific journal), ISSN 0147-5185

Invasive intraductal papillary-mucinous carcinoma (I-IPMC) is a heterogeneous entity with various postoperative outcomes. The aim of this study is to characterize early-stage I-IPMC with nonaggressive characteristics. One hundred and four patients with intraductal papillary-mucinous neoplasm (IPMN) were clinicopathologically investigated. The lesions were classified into 53 noninvasive IPMNs (adenoma, borderline, and,noninvasive IPMC) and 51 I-IPMCs on the basis of the WHO classification. I-IPMCs were divided further into 26 minimally invasive IPMCs (MI-IPMCs) and 25 invasive carcinomas originating in IPMC (IC-IPMCs) by new diagnostic criteria proposed in this study. We examined invasiveness of I-IPMC on 4 patterns, and defined simple and practical diagnostic criteria of minimal invasion for each invasive pattern. The disease-specific survival rates after 3, 5, and 10 years were 100%, 100%, and 100% for both noninvasive IPMN and MI-IPMC, and 51%, 38%, and 0% for IC-IPMC. The overall and disease-specific survival rates for MI-IPMC were both significantly better than those for IC-IPMC (P < 0.001), but there was no significant difference between noninvasive IPMN and MI-IPMC. Multivariate analysis showed that the factors indicative of poor prognosis were a diagnosis of I-IPMC classified as IC-IPMC and a high level of serum carbohydrate antigen 19-9. The prognosis of IC-IPMC was not significantly different from that of pancreatic ductal carcinoma in each of the corresponding tumor-node-metastasis stages. These findings suggest that a category of MI-IPMC provides more accurate and useful information of the stage and the aggressiveness of I-IPMC.

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A new formula for predicting liver metastasis in patients with colorectal cancer: Immunohistochemical analysis of a large series of 439 surgically resected cases

Ochiai, Hiroki, Nakanishi, Yukihiro, Fukasawa, Yuri, Sato, Yasunori, Yoshimura, Kimio, Moriya, Yoshihiro, Kanai, Yae, Watanabe, Masahiko, Hasegawa, Hirotoshi, Kitagawa, Yuko, Kitajima, Masaki, Hirohashi, Setsuo

ONCOLOGY (KARGER) 75 ( 1-2 ) 32 - 41 2008

Research paper (scientific journal), ISSN 0030-2414

Objective: The purpose of this study was to establish a new formula predicting liver metastasis in patients with colorectal cancer (CRC). Methods: Nine previously reported predictive markers for liver metastasis and/or prognosis (COX-2, dysadherin, E-cadherin, beta-catenin, Ki-67, p53, laminin5 gamma 2, matrilysin and MUC-1) were immunohistochemically investigated in 439 consecutive patients with CRC. We tried to determine the combination of molecules which best predicted liver metastasis. A formula for predicting liver metastasis was constructed using a training cohort comprising 150 cases, and applied to a validation cohort comprising 190 cases and another comprising 99 cases from an outside hospital. Results: A combination of dysadherin, E-cadherin and matrilysin was identified to be best for predicting liver metastasis (area under the curve value, 0.807). The predictive formula: 3 x dysadherin score [0 for low expression (<= 50% of tumor cells positive) or 1 for high expression (> 50%)] + 4 x E-cadherin score [0 for preserved (> 80% of tumor cells positive) or 1 for reduced (<= 80%)] + 2 x matrilysin score [0 for low expression (<= 30% of tumor cells positive) or 1 for high expression (> 30%)] was able to discriminate patients with liver metastasis in the training cohort with a sensitivity of 85.7% and a specificity of 58.9%. The discriminative capacity of the formula was validated in the first cohort with a sensitivity of 87.0% and a specificity of 66.5%, and in the second cohort with a sensitivity of 80% and a specificity of 60.0%. Conclusions: We have established a formula for predicting liver metastasis in patients with CRC, and confirmed that it has a high sensitivity potentially useful for clinical application. Copyright (C) 2008 S. Karger AG, Basel.

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A Novel Diagnostic Biomaker, LMP2, for Human Uterine Leiomyosarcoma

Hayashi, Takuma, Kobayashi, Yukihiro, Horiuchi, Akiko, Hiraoka, Nobuyoshi, Kanai, Yae, Konishi, Ikuo, Sano, Kenji

TUMOR BIOLOGY (KARGER) 29 47 - 47 2008

Research paper (scientific journal), ISSN 1010-4283

Alterations of DNA methylation associated with abnormalities of DNA methyltransferases in human cancers during transition from a precancerous to a malignant state

Kanai, Yae, Hirohashi, Setsuo

CARCINOGENESIS (OXFORD UNIV PRESS) 28 ( 12 ) 2434 - 2442 2007.12

Research paper (scientific journal), ISSN 0143-3334

Alterations of DNA methylation are one of the most consistent epigenetic changes in human cancers. Human cancers generally show global DNA hypomethylation accompanied by region-specific hypermethylation. Alterations of DNA methylation may result in chromosomal instability as a result of changes in chromatin structure. DNA hypermethylation of CpG islands silences various tumor-related genes. Alterations of DNA methylation are frequently observed in cancers associated with chronic inflammation and/or persistent infection with viruses or other pathogenic microorganisms, such as hepatitis B or C viruses, Epstein-Barr virus, human papillomavirus and Helicobacter pylori, or with cigarette smoking. Accumulating evidence suggests that alterations of DNA methylation are involved even in the early and precancerous stages. On the other hand, in patients with cancers, aberrant DNA methylation is significantly associated with poorer tumor differentiation, tumor aggressiveness and poor prognosis. Precancerous conditions showing alterations of DNA methylation may progress rapidly and generate more malignant cancers. DNA methyltransferase (DNMT) 1 over-expression is not a secondary result of increased cell proliferative activity but is significantly correlated with the CpG island methylator phenotype, which is defined as frequent DNA hypermethylation of C-type CpG islands that are usually methylated in a cancer-specific (not age-dependent) manner. Splicing alteration of DNMT3b may result in chromosomal instability through DNA hypomethylation of pericentromeric satellite regions. Alteration of DNA methylation may become an indicator for carcinogenetic risk estimation and early diagnosis of cancers and a biological predictor of poor prognosis in patients with cancers. Correction of DNA methylation status may offer a new strategy for prevention and therapy of cancers.

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Molecular markers and changes of computed tomography appearance in lung adenocarcinoma with ground-glass opacity

Yoshida, Yukihiro, Kokubu, Akiko, Suzuki, Kenji, Kuribayashi, Hidehiko, Tsuta, Koji, Matsuno, Yoshihiro, Kusumoto, Masahiko, Kanai, Yae, Asamura, Hisao, Hirohashi, Setsuo, Shibata, Tatsuhiro

JAPANESE JOURNAL OF CLINICAL ONCOLOGY (OXFORD UNIV PRESS) 37 ( 12 ) 907 - 912 2007.12

Research paper (scientific journal), ISSN 0368-2811

Background: High-resolution computed tomography (HRCT) of lung adenocarcinoma at early stage shows pure ground-glass opacity (GGO) and most cases of pure GGO remain stable during follow-up. There is no consensus on the strategy for follow-up. Identification of the molecular mechanisms that are associated with the natural history of lung adenocarcinoma should provide useful information.
Methods: Twenty-three lung adenocarcinomas that were followed-up for more than 6 months pre-operatively by HRCT were included in this study. Patterns of radiological changes during the follow-up period were classified into three groups; type 1, pure GGO without consolidation; type 2, appearance or increase in consolidation within pure GGO; type 3, consolidation without pure GGO. Mutational analysis of the epidermal growth factor receptor (EGFR) and K-ras genes and immunohistochemical staining of p53 protein were performed.
Results: EGFR mutations were found in 17 cases (74%), and there was no K-ras mutation. Positive staining of p53 was found in 8 cases (35%). As for radiological findings during the follow-up period, the frequencies of EGFR mutations and positive p53 staining were 67 and 0% in type 1 (n = 9), 89 and 44% in type 2 (n = 9) and 60 and 80% in type 3 (n = 5).
Conclusions: EGFR mutations were frequently found in lung adenocarcinoma with GGO on HRCT in this study. Inactivation of p53 may be associated with the appearance of central consolidation within pure GGO on HRCT which reflects invasive features and may be useful as a molecular marker during the follow-up of pure GGO.

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Invasive biliary cystic tumor without ovarian-like stroma

Ishibashi, Yuji, Ojima, Hidenori, Hiraoka, Nobuyoshi, Sano, Tsuyoshi, Kosuge, Tomoo, Kanai, Yae

PATHOLOGY INTERNATIONAL (BLACKWELL PUBLISHING) 57 ( 12 ) 794 - 798 2007.12

Research paper (scientific journal), ISSN 1320-5463

Presented herein is a rare case of invasive biliary cystic tumor without an ovarian-like stroma, and the apparent sequence underlying its malignant transformation, which was identified on detailed histological examination. A 54-year-old woman was incidentally diagnosed as having a cystic tumor in segment VIII of the liver, and central biseg-mentectomy was performed. Macroscopically the tumor measured 4.6 x 3.5 cm; and unilocular cystic and solid areas were seen on cut surface. Microscopically the tumor showed three types of neoplasia: adenoma and tubulopapillary adenocarcinoma in the cystic area, and invasive adenocarcinoma in the solid area. The relative area of the tumor occupied by each of these histological types was approximately 3%, 50% and 47%, respectively. Moreover, transitional zones between adenoma and tubulopapillary adenocarcinoma, and between tubulopapillary adenocarcinoma and invasive adenocarcinoma were noted. The immunohistochemical expression of Ki-67 and p53 increased gradually from adenoma through to tubulopapillary adenocarcinoma, and was highest in invasive adenocarcinoma. MUC1 was positive, and MUC2 and MUC5AC were both negative. No ovarian-like stroma or communication with the bile ducts around the tumor was found in any area of the specimen. On the basis of the World Health Organization histological classification and these pathological findings, the present case was diagnosed as invasive-type biliary cystadenocarcinoma.

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Sensitive detection of FGFR3 mutations in bladder cancer and urine sediments by peptide nucleic acid-mediated real-time PCR clamping

Miyake, Makito, Sugano, Kokichi, Kawashima, Kiyotaka, Ichikawa, Hiroki, Hirabayashi, Kaoru, Kodama, Tetsuro, Fujimoto, Hiroyuki, Kakizoe, Tadao, Kanai, Yae, Fujimoto, Kiyohide, Hirao, Yoshihiko

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (ACADEMIC PRESS INC ELSEVIER SCIENCE) 362 ( 4 ) 865 - 871 2007.11

Research paper (scientific journal), ISSN 0006-291X

Somatic mutations of the fibroblast growth factor receptor 3 (FGFR3) gene were detected by peptide nucleic acid (PNA)-mediated real-time PCR clamping. Mutation was detected in negative control containing only wild-type DNA due to a misincorporation of dNTPs to PNA binding sites when the amount of template DNA was decreased to 1 ng. Thus, the amount of template DNA was critical determinant of the assay sensitivity in PNA-mediated PCR clamping. Assay conditions were optimized to detect FGFR3 mutations in exons 7, 10, and 15, at a concentration of more than 1% mutated DNA using 50 ng of genomic DNA as the template. Mutations were detected in 12 of 13 (92.3%) tumor tissues and 11 of 13 (84.6%) urine samples from patients with superficial bladder cancer, while no mutations were detected in tissues and/or urine samples from patients with muscle-invasive bladder cancer or chronic cystitis. (c) 2007 Elsevier Inc. All rights reserved.

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Promoter hypermethylation contributes to frequent inactivation of a putative conditional tumor suppressor gene connective tissue growth factor in ovarian cancer

Kikuchi, Ryoko, Tsuda, Hitoshi, Kanai, Yae, Kasamatsu, Takahiro, Sengoku, Kazuo, Hirohashi, Setsuo, Inazawa, Johji, Imoto, Issei

CANCER RESEARCH (AMER ASSOC CANCER RESEARCH) 67 ( 15 ) 7095 - 7105 2007.08

Research paper (scientific journal), ISSN 0008-5472

Connective tissue growth factor (CTGF) is a secreted protein belonging to the CCN family, members of which are implicated in various biological processes. We identified a homozygous loss of CTGF (6q23.2) in the course of screening a panel of ovarian cancer cell lines for genomic copy number aberrations using in-house array-based comparative genomic hybridization. CTGF mRNA expression was observed in normal ovarian tissue and immortalized ovarian epithelial cells but was reduced in many ovarian cancer cell lines without its homozygous deletion (12 of 23 lines) and restored after treatment with 5-aza 2 '-deoxycytidine. The methylation status around the CTGF CpG island correlated inversely with the expression, and a putative target region for methylation showed promoter activity. CTGF methylation was frequently observed in primary ovarian cancer tissues (39 of 66, 59%) and inversely correlated with CTGF mRNA expression. In an immunohistochemical analysis of primary ovarian cancers, CTGF protein expression was frequently reduced (84 of 103 cases, 82%). Ovarian cancer tended to lack CTGF expression more frequently in the earlier stages (stages I and II) than the advanced stages (stages III and IV). CTGF protein was also differentially expressed among histologic subtypes. Exogenous restoration of CTGF expression or treatment with recombinant CTGF inhibited the growth of ovarian cancer cells lacking its expression, whereas knockdown of endogenous CTGF accelerated growth of ovarian cancer cells with expression of this gene. These results suggest that epigenetic silencing by hypermethylation of the CTGF promoter leads to a loss of CTGF function, which may be a factor in the carcinogenesis of ovarian cancer in a stage-dependent and/or histologic subtype-dependent manner.

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肝多段階発がん過程におけるFOXP3+制御性T細胞について(FOXP3+ Regulatory T Cells Affect the Development and Progression of Hepatocarcinogenesis)

小林規俊, 平岡伸介, 山上亘, 尾島英知, 金井弥栄, 小菅智男, 中島淳, 廣橋説雄

日本癌学会総会記事 ((一社)日本癌学会) 66回 42 - 42 2007.08

ISSN 0546-0476

Increased expression of DNA methyltransferase 1 (DNMT1) protein in uterine cervix squamous cell carcinoma and its precursor lesion

Sawada, Morio, Kanai, Yae, Arai, Eri, Ushijima, Saori, Ojima, Hidenori, Hirohashi, Setsuo

CANCER LETTERS (ELSEVIER IRELAND LTD) 251 ( 2 ) 211 - 219 2007.06

Research paper (scientific journal), ISSN 0304-3835

Aberrant DNA methylation has been shown to play important roles during multistage carcinogenesis in various human organs. The aim of this study was to evaluate the significance of DNA methyltransferase 1 (DNMT1) protein expression during cervical carcinogenesis. We carried out an immunohistochemical examination for DNMT1 in 34 samples of histologically normal squamous epithelium, 36 samples of low-grade cervical intraepithelial neoplasia (CIN), 61 samples of higher-grade CIN and 30 samples of squamous cell carcinoma of the uterine cervix. The DNMT1 protein expression score, reflecting the intensity and incidence of DNMT1 nuclear immunoreactivity, was increased even in low-grade CIN (P < 0.0001) in comparison with histologically normal squamous epithelium and was further increased in higher-grade CIN (P < 0.0001 compared to low-grade CIN). The DNMT1 protein expression score remained at a plateau in microinvasive carcinoma (Stage IA, P = 0.0690 compared to higher-grade CIN) and then decreased with cancer invasion (Stage IB or more, P = 0.0 176 compared to Stage IA), whereas the proliferating cell nuclear antigen (PCNA) labeling index did not decrease with cancer invasion (P = 0.8259 between Stage IA and Stage IB or more). Thus, the DNMT1 protein expression score and the PCNA labeling index were not mutually correlated in squamous cell carcinoma of the uterine cervix (P = 0.2304). These data suggest that progressively increasing expression of DNMT1 protein is not entirely a secondary result of increased cell proliferative activity, but is associated with an early step of multistage cervical carcinogenesis. (C) 2006 Elsevier Ireland Ltd. All rights reserved.

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

Loukopoulos, Panayiotis, Shibata, Tatsuhiro, Katoh, Hiroto, Kokubu, Akiko, Sakamoto, Michiie, Yamazaki, Ken, Kosuge, Tomoo, Kanai, Yae, Hosoda, Fumie, Imoto, Issei, Ohki, Misao, Inazawa, Jyoji, Hirohashi, Setsuo

CANCER SCIENCE (BLACKWELL PUBLISHING) 98 ( 3 ) 392 - 400 2007.03

Research paper (scientific journal), ISSN 1347-9032

We analyzed the subchromosomal numerical aberrations of 44 surgically resected pancreatic adenocarcinomas by array-based comparative genomic hybridization. The aberration profile ranged widely between cases, suggesting the presence of multiple or complementary mechanisms of evolution in pancreatic cancer, and was associated with lymph node metastasis and venous or serosal invasion. A large number of small loci, previously uncharacterized in pancreatic cancer, showed non-random loss or gain. Frequent losses at 1p36, 4p16, 7q36, 9q34, 11p15, 11q13, 14q32-33, 16p13, 17p11-13, 17q11-25, 18q21-tel, 19p13, 21q22 and 22q11-12, and gains at 1q25, 2p16, 2q21-37, 3q25, 5p14, 5q11-13, 7q21, 7p22, 8p22, 8q21-23, 10q21, 12p13, 13q22, 15q13-22 and 18q11 were identified. Sixteen loci were amplified recurrently. We identified novel chromosomal alterations that were significantly associated with a range of malignant phenotypes. Gain of LUNX, HCK, E2F1 and DNMT3b at 20q11, loss of p73 at 1p36 and gain of PPM1D at 17q23 independently predicted patient outcome. Expression profiling of amplified genes identified Smurf1 and TRRAP at 7q22.1, BCAS1 at 20q13.2-3, and VCL at 10q22.1 as potential novel oncogenes. Our results contribute to a complete description of genomic structural aberrations and the identification of potential therapeutic targets and genetic indicators that predict patient outcome in pancreatic adenocarcinoma.

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FOXP3(+) regulatory T cells affect the development and progression of hepatocarcinogenesis

Kobayashi, Noritoshi, Hiraoka, Nobuyoshi, Yamagami, Wataru, Ojima, Hidenori, Kanai, Yae, Kosuge, Tomoo, Nakajima, Atsushi, Hirohashi, Setsuo

CLINICAL CANCER RESEARCH (AMER ASSOC CANCER RESEARCH) 13 ( 3 ) 902 - 911 2007.02

Research paper (scientific journal), ISSN 1078-0432

Purpose: Tumor-infiltrating lymphocytes represent the host immune response to cancer. CD4(+) CD25(+) FOXP3(+) regulatory T cells (Tregs) suppress the immune reaction. The aim of the present study was to investigate the clinicopathologic significance and roles of Tregs and CD8(+) T cells during hepatocarcinogenesis.
Experimental Design: We examined the infiltration of FOXP3(+) Tregs and CD8(+) Tcells in the tumor stroma and nontumorous liver parenchyma using 323 hepatic nodules including precursor lesions, early hepatocellular carcinoma (HCC), and advanced HCC, along with 39 intrahepatic cholangiocarcinomas and 59 metastatic liver adenocarcinomas. We did immunohistochemical comparative studies.
Results: The prevalence of Tregs was significantly higher in HCC than in the nontumorous liver (P < 0.001). The patient group with a high prevalence of Tregs infiltrating HCC showed a significantly lower survival rate (P = 0.007). Multivariate analysis revealed that the prevalence of Tregs infiltrating HCC was an independent prognostic factor. The prevalence of Tregs increased in a stepwise manner (P < 0.001) and that of CD8(+) T cells decreased during the progression of hepatocarcinogenesis (P < 0.001). Regardless of the presence of hepatitis virus infection or histopathologic evidence of hepatitis, the prevalence of Tregs was significantly increased in nontumorous liver bearing primary hepatic tumors.
Conclusions: Tregs play a role in controlling the immune response to HCC during the progression of hepatocarcinogenesis. It has been suggested that primary hepatic cancers develop in liver that is immunosuppressed by a marked infiltration of Tregs. A high prevalence of Tregs infiltrating HCC is thought to be an unfavorable prognostic indicator.

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Prognostic value of tumor architecture, tumor-associated vascular characteristics, and expression of angiogenic molecules in pancreatic endocrine tumors

Takahashi, Yu, Akishima-Fukasawa, Yuri, Kobayashi, Noritoshi, Sano, Tsuyoshi, Kosuge, Tomoo, Nimura, Yuji, Kanai, Yae, Hiraoka, Nobuyoshi

CLINICAL CANCER RESEARCH (AMER ASSOC CANCER RESEARCH) 13 ( 1 ) 187 - 196 2007.01

Research paper (scientific journal), ISSN 1078-0432

Purpose: It is difficult to predict the biological behavior of pancreatic endocrine tumors (PETs). Our aim was to evaluate the prognostic significance of certain variables in PETs.
Experimental Design: The following variables were examined in 37 patients with PETs and then compared with other clinicopathologic characteristics: histologic tumor structure; microvessel density (MVD) measured by three different methods, including a unique method involving calculation of solid area MVD; endothelial proliferation; and the immunohistochemical expression of vascular endothelial growth factor-A and CXC chemokine CXCL-12. Intratumoral vascular structures were analyzed by double immunofluorescence using 30-mu m-thick sections.
Results: The presence of focal and intensive solid growth of tumor cells (large solid nests; P=0.003), low solid area MVD (P=0.002), a high endothelial cell proliferation index (EPI; P=0.005), and high expression of CXCL-12 in PET cells (P=0.018) were significant unfavorable prognostic indicators. The predominant structure of the overall tumor histology and the expression of vascular endothelial growth factor-A did not separate aggressive PETs. In areas of focal solid growth, tumor-associated blood vessels had obviously low MVD and high EPI, and their structures were poorly formed with highly abnormal features, in comparison with other areas. High expression of CXCL-12 in tumor cells was significantly associated with variables representing tumor growth, hematogenous tumor spread, low MVD, high EPI, and the presence of large solid nests.
Conclusions: This study has provided novel findings on the prognostic features of tumor architecture and tumor-associated angiogenesis in PETs. CXCL-12 is the first candidate molecule in association with neoangiogenesis in PETs.

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Primary adenocarcinoma of the rete testis with preceding diagnosis of pulmonary metastases

Nakagawa, Tohru, Hiraoka, Nobuyoshi, Ihara, Fumie, Komiyama, Motokiyo, Kanai, Yae, Matsuno, Yoshihiro

INTERNATIONAL JOURNAL OF UROLOGY (BLACKWELL PUBLISHING) 13 ( 12 ) 1532 - 1535 2006.12

Research paper (scientific journal), ISSN 0919-8172

We report a case of primary adenocarcinoma of the rete testis in a 55-year-old man with pulmonary metastases that were detected 11 months prior to the diagnosis of the primary lesion. Primary adenocarcinoma of the rete testis is an extremely rare malignant tumor with a poor outcome. The most common primary symptom is a scrotal mass, often accompanied by hydrocele and chronic epididymitis. The diagnosis is often delayed because of non-specific clinical presentation and symptoms. We cannot forget that rete testis is a possible primary site for a primary, unknown metastatic adenocarcinoma.

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Genetic inactivation of the APC gene contributes to the malignant progression of sporadic hepatocellular carcinoma: A case report

Katoh, Hiroto, Shibata, Tatsuhiro, Kokubu, Akiko, Ojima, Hidenori, Kosuge, Tomoo, Kanai, Yae, Hirohashi, Setsuo

GENES CHROMOSOMES & CANCER (WILEY-LISS) 45 ( 11 ) 1050 - 1057 2006.11

Research paper (scientific journal), ISSN 1045-2257

Although activated Wnt/beta-catenin pathway is considered to be one of the main driving forces of hepatocarcinogenesis, no somatic mutations of the adenomatous polyposis coli (APC) gene have been found in sporadic hepatocellular carcinoma (HCC) to date. Here we present a case of a sporadic nodule-in-nodule-type HCC that provides the first evidence that biallelic genetic inactivation of the APC gene contributed to the development of the tumor. High-density array-based comparative genomic hybridization (aCGH) was performed to clarify genome-wide chromosomal structural alteration profiles of both early and advanced components of this nodule-in-nodule HCC. aCGH analysis revealed a chromosomal loss of the AIPC gene locus only in the inner advanced component of this tumor. Direct sequencing of the remaining allele revealed a nonsense mutation at codon 682 in the Armadillo repeats, resulting in a truncated protein that lacked all of the beta-catenin-binding motifs. Nonsense mutations at this location are rare among other types of cancer. In conclusion, combined with an immunohistochemical analysis of the beta-catenin protein, this case provides the first evidence that genetic inactivation of the APC gene can play a significant role in the progression of sporadic HCC, probably by activating the Wnt/beta-catenin pathway. (c) 2006 Wiley-Liss, Inc.

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Expression status of RUNX1/AML1 in normal gastric epithelium and its mutational analysis in microdissected gastric cancer cells

Usui, Takebumi, Aoyagi, Kazuhiko, Saeki, Norihisa, Nakanishi, Yukihiro, Kanai, Yae, Ohki, Misao, Ogawa, Kenji, Yoshida, Teruhiko, Sasaki, Hiroki

INTERNATIONAL JOURNAL OF ONCOLOGY (PROFESSOR D A SPANDIDOS) 29 ( 4 ) 779 - 784 2006.10

Research paper (scientific journal), ISSN 1019-6439

Although Runt-related transcription factors RUNXs (RUNX1-3) have a high similarity in their structure, only RUNX3 is known to be involved in gastric carcinogenesis. First, we examined mRNA expression of these three RUNX genes in the gastric mucosa, and, finding only RUNX2 was not expressed there, we further investigated RUNX1 and RUNX3 expression in three regions including the pit, isthmus/ neck, and gland regions of the human normal stomach and whether RUNX1 is involved in gastric carcinogenesis. The mRNA expression of RUNX1 and RUAX3 was examined by use of the three regions isolated by laser-captured microdissection (LCM) and by use of primary gastric cancer tissues. Furthermore, RUNX1 mutational analysis was performed in the cancer cells, which also were isolated from 44 paraffin-embedded gastric cancer tissues by LCM. RUNX1 was coexpressed with RUNX3 in the pit region, and has cell growth-inhibition activity similar to RUNX3. RUNX3 has been reported to be suppressed by DNA methylation in a subset of gastric cancers; however, the expression of RUNX1 mRNA was observed in all of the gastric cancer cell lines and gastric cancer tissues that we examined. No RUNX1 mutation was found in the 44 gastric cancer patients. Although RUNX1 is similar to RUNX3 in both the expression pattern in the stomach and its cell growth-inhibition activity, RUNX1 is not involved in most cases of gastric cancers. These results suggest that the transcriptional target genes are different between these two family genes.

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DNA methylation of multiple tumor-related genes in association with overexpression of DNA methyltransferase 1 (DNMT1) during multistage carcinogenesis of the pancreas

Peng, Dun-Fa, Kanai, Yae, Sawada, Morio, Ushijima, Saori, Hiraoka, Nobuyoshi, Kitazawa, Sohei, Hirohashi, Setsuo

CARCINOGENESIS (OXFORD UNIV PRESS) 27 ( 6 ) 1160 - 1168 2006.06

Research paper (scientific journal), ISSN 0143-3334

To evaluate the significance of alterations in DNA methylation during multistage carcinogenesis of the pancreas, tissue samples of 13 peripheral pancreatic duct epithelia showing no remarkable histological changes without inflammatory background (DE), 20 peripheral pancreatic duct epithelia showing no remarkable histological changes with inflammatory background (DEI), 40 pancreatic intraepithelial neoplasias (PanIN) and 147 areas of ductal carcinoma were microdissected from surgically resected specimens from 58 patients and were embedded into agarose beads. The embedded tissue samples were subjected to methylation-specific PCR (MSP) to evaluate the DNA methylation status of the p14, p15, p16, p73, APC, hMLH1, MGMT, BRCA1, GSTPI, TIMP-3, CDH1 and DAPK-1 genes. The prevalence of DNA methylation of at least one of the 12 genes and the average number of methylated genes were significantly higher in both DEI (60% and 0.85 +/- 0.88, P = 0.0151 and P = 0.0224, respectively) and PanIN (67.5% and 0.95 +/- 0.85, P = 0.0014 and P = 0.0028, respectively) than in DE (15.4% and 0.15 +/- 0.38), and were further increased in ductal carcinoma (98.3% and 2.50 +/- 1.35, P < 0.0001 and P < 0.0001, respectively). The BRCA1, APC, p16 and TIMP-3 genes were frequently methylated in ductal carcinoma (60.3, 58.6, 39.3 and 30.9%, respectively). Considerable heterogeneity of DNA methylation status was observed among multiple microdissected areas from individual ductal carcinomas, and the number of methylated genes per area was significantly correlated with poorer tumor differentiation (P = 0.0249). The average number of methylated genes in ductal carcinomas was significantly correlated with DNMT1 protein expression level (P = 0.0093). These data suggest that accumulation of DNA methylation of multiple tumor-related genes is involved in multistage carcinogenesis of the pancreas from early precancerous stages to malignant progression and that DNMT1 protein overexpression may be responsible for this aberrant DNA methylation.

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Regional DNA hypermethylation and DNA methyltransferase (DNMT) 1 protein overexpression in both renal tumors and corresponding nontumorous renal tissues.

Arai E, Kanai Y, Ushijima S, Fujimoto H, Mukai K, Hirohashi S

Int J Cancer (WILEY-LISS) 119 ( 2 ) 288 - 296 2006

Accepted, ISSN 00207136

To evaluate the significance of altered DNA methylation during renal tumorigenesis, tumorous tissues (T) and corresponding nontumorous renal tissues (N) from 94 patients with renal tumors, and normal renal tissues (C) from 16 patients without renal tumors were investigated. DNA methylation status on CpG islands of the p16, human MutL homologue 1 (hMLHI), von-Hippel Lindau (VHL) and thrombospondin-I (THBS-1) genes and the methylated in tumor (MINT) -1, -2, -12, -25 and -31 clones and DNA methyltransferase (DNMT) 1 expression were examined by bisulfite modification and immunohistochemistry, respectively. The average number of methylated CpG islands was significantly higher in N than in C, and was even higher in T. The average number of methylated CpG islands in N was significantly correlated with a higher histological grade of corresponding conventional renal cell carcinomas (RCCs). The average number of methylated CpG islands in RCCs was significantly correlated with macroscopic configuration with extratiodular or multinodular growth, higher histological grade, infiltrating growth pattern and vascular involvement. The recurrence-free survival rate of patients with RCCs showing accumulation of DNA methylation was significantly lower than that of patients not showing this feature. The incidence of nuclear immunoreactivity for DNMT1 tended to be higher in proximal tubules from N than in those from C, and was significantly higher in RCCs. From the viewpoint of altered DNA methylation, N is at the precancerous stage, and N showing accumulation of DNA methylation may generate more malignant RCCs. Regional DNA hypermethylation may be associated with renal tumorigenesis from a precancerous condition to malignant progression and become a predictor of patient prognosis. (c) 2006 Wiley-Liss, Inc.

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Leiomyosarcoma of the renal vein: arterial encasement on contrast-enhanced dynamic computed tomography.

Maeda T, Tateishi U, Fujimoto H, Kanai Y, Sugimura K, Arai Y

Int J Urol (BLACKWELL PUBLISHING) 13 ( 5 ) 611 - 612 2006

Accepted, ISSN 09198172

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Solid-pseudopapillary neoplasms of the pancreas in men and women: do they differ?

Takahashi Y, Hiraoka N, Onozato K, Shibata T, Kosuge T, Nimura Y, Kanai Y, Hirohashi S

Virchows Arch (SPRINGER) 448 ( 5 ) 561 - 569 2006

Accepted, ISSN 09456317

Solid-pseudopapillary neoplasms (SPNs) of the pancreas are uncommon and occur preferentially in young women. The question whether the features of SPNs occurring in men differ from those in women has not yet been studied. For a better understanding of the clinicopathological features of SPNs of both sexes, we studied a series of 14 tumors surgically resected at a Japanese hospital within a period of 14 years. This series was composed of seven men and seven women. All these SPNs demonstrated nuclear and cytoplasmic accumulation of beta-catenin protein in immunohistochemistry and 86% of them had activating mutations of beta-catenin gene. No pancreatic neuroendocrine tumors showed such immunohistochemical findings and genetic alterations. In our series, most SPNs in women showed encapsulation by thick fibrous tissue and massive degenerative changes. Most SPNs in men exhibited solid components without prominent degenerative changes, even though they were of a similar size to those in women. These findings suggest that SPNs in men tend to be a solid mass with slower progression of degenerative changes during their growth compared to that in women. Nuclear accumulation of beta-catenin appears to be a useful marker of SPN, which allows male SPNs to be correctly diagnosed despite their less typical features.

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Epigenetic instability and chromosomal instability in hepatocellular carcinoma.

Katoh H, Shibata T, Kokubu A, Ojima H, Fukayama M, Kanai Y, Hirohashi S

Am J Pathol (AMER SOC INVESTIGATIVE PATHOLOGY, INC) 168 ( 4 ) 1375 - 1384 2006

Accepted, ISSN 00029440

The aim of this study was to clarify the association between the epigenetic instability phenotype and the chromosomal instability phenotype in primary hepatocellular carcinoma (HCC). Sixty primary HCC tumors were examined. Methylation status for nine CpG islands (the p16, COX2, GSTP1, RASSF1A, E-cadherin, and APC gene promoters, and the MINT 1, 25, and 31 clones) was evaluated by methylation-specific polymerase chain reaction. Chromosomal structural alterations of these 60 HCC tumors were characterized in our previous study by using whole genomic array-based comparative genomic hybridization. We found that the epigenetic instability phenotype and the chromosomal instability phenotype are not mutually exclusive in hepatocarcinogenesis and that they do not show a simple cause-and-effect relationship. Hepatitis virus infection in the background liver was significantly associated with these instability phenotypes. Furthermore, we identified an epigenetic instability-dependent HCC that shows frequent epigenetic aberrations without chromosomal instability. it was noteworthy that epigenetic instability-positive and -negative HCCs displayed distinctive combinations of chromosomal structural alterations. In summary, by combined analyses of genetic and epigenetic aberration profiles in HCC, we obtained a comprehensive view of genomic alterations in hepatocarcinogenesis. Our results have clinical relevance because epigenetic instability-dependent HCCs may respond well to methylation inhibitory therapies.

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Promoter hypermethylation of the potential tumor suppressor DAL-1/4.1B gene in renal clear cell carcinoma

Yamada D, Kikuchi S, Williams YN, Sakurai-Yageta M, Masuda M, Maruyama T, Tomita K, Gutmann DH, Kakizoe T, Kitamura T, Kanai Y, Murakami Y

Int J Cancer (WILEY-LISS) 118 ( 4 ) 916 - 923 2006

Accepted, ISSN 00207136

Renal clear cell carcinoma (RCCC) is a malignant tumor with poor prognosis caused by the high incidence of metastasis to distal organs. Although metastatic RCCC cells frequently show aberrant cytoskeletal organization, the underlying mechanism has not been elucidated. DAL-1/4.1B is an actin-binding protein implicated in the cytoskeleton-associated processes, while its inactivation is frequently observed. in lung and breast cancers and meningiomas, suggesting that 4.1B is a potential tumor suppressor. We studied a possible involvement of 4.1B in RCCCs and evaluated it as a clinical indicator. 4.1B protein was detected in the proximal convuluted tubules of human kidney, the presumed cell of origin of RCCC. On the other hand, loss or marked reduction of its expression was observed in 10 of 19 (53%) renal cell carcinoma (RCC) cells and 12 of 19 (63%). surgically resected RCCC by reverse transcription-PCR. Bisulfite sequencing or bisulfite SSCP analyses revealed that the 4.1B promoter was methylated in 9 of 19 (47%) RCC cells and 25 of 55 (45%) surgically resected RCCC, and inversely correlated with 4.1B expression (p < 0.0001). Aberrant methylation appeared to be a relatively early event because more than 40% of the tumors with pT1a showed hypermethylation. Furthermore, 4.1B methylation correlated with a nuclear grade (p = 0.017) and a recurrence-free survival (p = 0.0036) and provided an independent prognostic factor (p = 0.038, relative risk 10.5). These results indicate that the promoter methylation of the 4.1B is one of the most frequent epigenetic alterations in RCCC and could predict the metastatic recurrence of the surgically resected RCCC. (c) 2005 Wiley-Liss, Inc.

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21 Role of Immunohistochemical Expression of DMA Methyltransferase 1 Protein in Gastric Carcinoma

Kanai Y., Arai E., Etoh T.

Handbook of Immunohistochemistry and in Situ Hybridization of Human Carcinomas (Handbook of Immunohistochemistry and in Situ Hybridization of Human Carcinomas) 4 257 - 261 2006

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DNA hypomethylation on pericentromeric satellite regions significantly correlates with loss of heterozygosity on chromosome 9 in urothelial carcinomas.

Nakagawa T, Kanai Y, Ushijima S, Kitamura T, Kakizoe T, Hirohashi S

J Urol (LIPPINCOTT WILLIAMS & WILKINS) 173 ( 1 ) 243 - 246 2005

Accepted, ISSN 00225347

Purpose: DNA methylation has important roles in genomic stability. Accordingly DNA hypomethylation on pericentromeric satellite regions may induce chromosomal instability through heterochromatin decondensation and chromosomal recombination enhancement. We elucidated the significance of aberrant DNA methylation on pericentromeric satellite regions during urothelial carcinogenesis.
Materials and Methods: We examined DNA methylation status on satellites 2 and 3 by Southern blotting and determined the allelic status of chromosome 9 using 6 microsatellite markers (D9S775, D9S925, D9S304, D9S303, D9S283 and D9S747) in 27 transitional cell carcinomas of the bladder, ureter or renal pelvis and corresponding noncancerous tissues.
Results: DNA hypomethylation on satellites 2 and 3 was detected in 2 (7%) and no (0%) noncancerous tissues, and in 11 (41%) and 12 (44%) urothelial carcinomas, respectively. DNA hypomethylation in urothelial carcinomas significantly correlated with histological grade (p = 0.0012 and 0.0043), invasion depth (p = 0.0055 and 0.0228) and morphological structure (papillary vs nodular, p = 0.0161 and 0.0297) for satellites 2 and 3, respectively. Loss of heterozygosity on at least 1 locus of chromosome 9 was detected in 14 urothelial carcinomas (52%). DNA hypomethylation on satellites 2 (p = 0.0098) and 3 (p = 0.0034) significantly correlated with loss of heterozygosity on chromosome 9.
Conclusions: DNA hypomethylation on pericentromeric satellite regions may participate in the development and progression of urothelial carcinomas by inducing loss of heterozygosity on chromosome 9.

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Clinicopathologic significance of dysadherin expression in cutaneous malignant melanoma: immunohistochemical analysis of 115 patients.

Nishizawa A, Nakanishi Y, Yoshimura K, Sasajima Y, Yamazaki N, Yamamoto A, Hanada K, Kanai Y, Hirohashi S.

Cancer (JOHN WILEY & SONS INC) 103 ( 8 ) 1693 - 1700 2005

Accepted, ISSN 0008543X

BACKGROUND. The E-cadherin-mediated cell adhesion system is frequeutly inactivated by multiple mechanisms and is involved in tumor progression in many types of cancer. Recently, the authors reported a novel cell membrane glycoprotein, dysadherin, which has an anti-cell-cell adhesion function and down-regulates E-cadherin.
METHODS. Expression of both dysadherin and E-cadherin was investigated immunohistochemically in 115 patients with cutaneous malignant melanoma to determine the correlation between the 2 molecules and their associations with both patient survival and the clinicopathologic features of the tumors.
RESULTS. Dysadherin and E-cadherin were expressed at the cell membranes of melanoma cells. Fifty-two percent of the tumors showed dysadherin immunopositivity, and 91% of the tumors showed reduced E-cadherin immunopositivity. There was no significant inverse correlation between dysadherin expression and E-cadherin expression. Increased dysadherin expression was significantly correlated with nodular subtype (P = 0.042), Clark level (P < 0.001), tumor thickness (P < 0.001), ulceration (P = 0.008), lymph node metastasis (P < 0.001), high TNM classification (P < 0.001), and poor patient survival (P < 0.001). Multivariate analysis of patient survival revealed that increased dysadherin expression was a significant predictor of poor Survival (P < 0.001).
CONCLUSIONS. Thus, increased expression of dysadherin was a significant indicator of poor prognosis in patients with cutaneous malignant melanoma. (c) 2005 American Cancer Society.

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Array-based comparative genomic hybridization analysis of high-grade neuroendocrine tumors of the lung.

Peng WX, Shibata T, Katoh H, Kokubu A, Matsuno Y, Asamura H, Tsuchiya R, Kanai Y, Hosoda F, Sakiyama T, Ohki M, Imoto I, Inazawa J, Hirohashi S.

Cancer Sci (BLACKWELL PUBLISHING) 96 ( 10 ) 661 - 667 2005

Accepted, ISSN 13479032

We examined a large number of primary high-grade neuroendocrine tumors of the lung (10 small cell lung carcinomas and 31 large cell neuroendocrine carcinomas) by using array-based comparative genomic hybridization using microarrays spotted with 800 bacterial artificial chromosome clones containing tumor-related genes from throughout the human genome. We identified the genome-wide copy number alteration profiles of these tumors, including recurrent amplifications located at 2q21.2, 3q21-27, 3q26, 3q27-29, 5p14.2, 5p13, 7q21.1, 8q21, and 8q24 and homozygous deletions at 1p36, 4p16, 4p16.3, 9p21.3, 9p21, 19p13.3, and 20q13. Our results revealed that small cell lung carcinomas and large cell neuroendocrine carcinomas have multiple characteristic chromosomal alterations in common, but that distinctive alterations also exist between the two subtypes. Moreover, we found that the two subtypes undergo different processes of accumulating these genetic alterations during tumor development. By comparing the genetic profiles with the clinicopathological features, we discovered many chromosomal loci whose alterations were significantly associated with clinical stage and patient prognosis. These results will be valuable for evaluating clinical status, including patient prognosis, and for identifying novel molecular targets for effective therapies.

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Mutations of the epidermal growth factor receptor gene in atypical adenomatous hyperplasia and bronchioloalveolar carcinoma of the lung.

Yoshida Y, Shibata T, Kokubu A, Tsuta K, Matsuno Y, Kanai Y, Asamura H, Tsuchiya R, Hirohashi S.

Lung Cancer (ELSEVIER IRELAND LTD) 50 ( 1 ) 1 - 8 2005

Accepted, ISSN 01695002

A hypothesis of multistep carcinogenesis of lung adenocarcinoma from atypical adenomatous hyperplasia (AAH) to invasive adenocarcinoma through bronchioloalveolar carcinoma (BAC) has been proposed. However, the genetic alterations that play a role during these processes are not yet clear. Recently, somatic mutations of the epidermal growth factor receptor (EGFR) gene were found in lung adenocarcinoma. We examined the status of EGFR mutations in AAH and BAC to elucidate the role they play during multistage of lung adenocarcinoma. We found somatic EGFR mutations in 3% (1/35) of AAH, 10.8% (4/37) of BAC and 41.9% (13/31) of invasive adenocarcinoma. Sixteen of 18 EGFR mutations were found in exon 19 and two were in exon 21. Among the 16 EGFR mutations in exon 19, 13 were deletions of 15 bp and one was an insertion/duplication of 18 bp. Mutations of the K-ras gene were detected in 26.7% (8/30) of AAH, 16.7% (5/30) of BAC and 10% (3/30) of invasive adenocarcinoma. None of the tumors with EGFR mutations had K-ras mutation simultaneously. Patients who had invasive adenocarcinoma with EGFR mutations were younger than those without mutations (60.6 versus 67.4 years, p = 0.03). These results suggest that tumors with EGFR mutations may progress more rapidly and develop into invasive cancer faster than those without mutations. Alternatively it is also possible that some invasive adenocarcinomas with EGFR mutations may not follow the AAH-adenocarcinoma sequence. We analyzed 24 patients with multiple lung lesions and 13 patients had at least one lesion that had either an EGFR or K-ras mutation. In all cases each lesion had a different mutation status. This finding suggests that the genetic alterations responsible for the development of lung adenocarcinoma occur randomly even under exposure to the same carcinogen. © 2005 Elsevier Ireland Ltd. All rights reserved.

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Genetic classification of lung adenocarcinoma based on array-based comparative genomic hybridization analysis: its association with clinicopathologic features.

Shibata T, Uryu S, Kokubu A, Hosoda F, Ohki M, Sakiyama T, Matsuno Y, Tsuchiya R, Kanai Y, Kondo T, Imoto I, Inazawa J, Hirohashi S.

Clin Cancer Res (AMER ASSOC CANCER RESEARCH) 11 ( 17 ) 6177 - 6185 2005

Accepted, ISSN 10780432

The array-based comparative genomic hybridization using microarrayed bacterial artificial chromosome clones allows high-resolution analysis of genome-wide copy number changes in tumors. To analyze the genetic alterations of primary lung adenocarcinoma in a high-throughput way, we used laser-capture microdissection of cancer cells and array comparative genomic hybridization focusing on 800 chromosomal loci containing cancer-related genes. We identified a large number of chromosomal numerical alterations, including frequent amplifications on 7p12, 11q13,12q14-15, and 17q21, and two homozygous deletions on 9p21 and one on 8p23. Unsupervised hierarchical clustering analysis of multiple alterations revealed three subgroups of lung adenocarcinoma that were characterized by the accumulation of distinct genetic alterations and associated with smoking history and gender. The mutation status of the epidermal growth factor receptor (EGFR) gene was significantly associated with specific genetic alterations and supervised clustering analysis based on EGFR gene mutations elucidated a subgroup including all EGFR gene mutated tumors, which showed significantly shorter disease-free survival. Our results suggest that there exist multiple molecular carcinogenesis pathways in lung adenocarcinoma that may associate with smoking habits and gender, and that genetic cancer profiling will reveal previously uncharacterized genetic heterogeneity of cancer and be beneficial in estimating patient prognosis and discovering novel cancer-related genes including therapeutic targets.

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Prognostic significance of tissue factor in pancreatic ductal adenocarcinoma.

Nitori N, Ino Y, Nakanishi Y, Yamada T, Honda K, Yanagihara K, Kosuge T, Kanai Y, Kitajima M, Hirohashi S.

Clin Cancer Res (AMER ASSOC CANCER RESEARCH) 11 ( 7 ) 2531 - 2539 2005

Accepted, ISSN 10780432

Tissue factor (TF) is a transmembrane glycoprotein that plays roles in the blood coagulation and intracellular signaling pathways, and has also been suggested to modulate the biological behavior of cancer cells. In order to examine the clinicopathologic significance of TF expression in pancreatic ductal adenocarcinoma, TF expression was determined by immunohistochemistry using a newly raised anti-TF monoclonal antibody in 113 patients who had undergone surgical resection of pancreatic ductal adenocarcinoma. According to the incidence of tumor cell immunopositivity, patients were divided into "negative TF (0%), "weak TF (<25%), or "high TF (25% or more) groups, which accounted for 11.6% (n = 13), 44.2% (n = 50), and 44.2% (n = 50) of the total, respectively. Increased TF expression was correlated with the extent of the primary tumor (P = 0.0043), lymph node metastasis (P = 0,0043), lymphatic distant metastasis (P 0.0039), advanced tumor-node-metastasis stage (P = 0.0002),and high tumor grade (P 0,0164). Multivariate analysis using the Cox proportional hazards model showed that high TF expression was an independent negative predictor for survival (hazard ratio, 2.014; P = 0.0076). Moreover, patients with TF-negative tumors had a significantly better prognosis even if lymph node metastasis was present (P < 0.0001). We also showed that TF knockdown by RNA interference suppressed the invasiveness of a pancreatic adenocarcinoma cell line in vitro. These results indicate that TF expression may contribute to the aggressiveness of pancreatic ductal adenocarcinoma by stimulating tumor invasiveness, and that evaluation of the primary tumor for TF expression may identify patients with a poor prognosis.

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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, Loukopoulos P, Kanai Y, Kosuge T, Fukayama M, Kondo T, Sakamoto M, Hosoda F, Ohki M, Imoto I, Inazawa J, Hirohashi S.

J Hepatol (ELSEVIER SCIENCE BV) 43 ( 5 ) 863 - 874 2005

ISSN 01688278

Background/Aims: We conducted an analysis of chromosomal numerical aberrations and their clinical significance in hepatocellular carcinoma.
Methods: We analyzed 87 hepatocellular carcinomas by array-based comparative genomic hybridization with an array containing 800 bacterial artificial chromosome clones.
Results: Frequent (> 30%) chromosomal losses on 1p36.1, 4q21-25, 4q34-35.1, 8p23.3b-11.1, 13q14.1-14.3, 16p13.3, 16q22.1-24.3b, 17p13.3-13.1 and 17p13.3-11, and gains on 1q21-44f, 2q21.2,2q34,3q11.2,5p14.2,5q13.2-14,7p22,7p14. 2, 7q21.1, 7q22.3, 7q34, 8q12-24.3 and 17q23, were observed. Recurrent (> 5%) amplifications were detected on 1q25, 8q11 and 11q11, and we discovered a novel homozygous deletion at 14q32.11. The extent of chromosomal aberrations correlated significantly with various clinicopathological characteristics of the tumors, and increased in a stepwise manner with the progression of hepatocellular carcinoma. We also identified novel chromosomal alterations that were significantly associated with a range of malignant phenotypes. Multivariate analysis revealed that both chromosomal loss on 17p13.3 and gain on 8q11 are independent prognostic indicators.
Conclusions: Our results contribute to a complete description of genomic structural aberrations in relation to hepatocarcinogenesis and provide a valuable basis from which we can begin to understand the characteristics of tumors, predict patient outcomes and discover novel therapeutic targets for hepatocellular carcinoma. (c) 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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Loss of blood group A antigen expression in bladder cancer caused by allelic loss and/or methylation of the ABO gene.

Chihara Y, Sugano K, Kobayashi A, Kanai Y, Yamamoto H, Nakazono M, Fujimoto H, Kakizoe T, Fujimoto K, Hirohashi S, Hirao Y.

Lab Invest (NATURE PUBLISHING GROUP) 85 ( 7 ) 895 - 907 2005

Accepted, ISSN 00236837

Loss of ABO blood group antigen expression has been reported in transitional cell carcinoma ( TCC) of the bladder. Synthesis of the ABO blood group antigen was genetically determined by allelic variants of the ABO gene assigned on 9q34.1. We analyzed loss of heterozygosity ( LOH) and promoter hypermethylation of the ABO gene in TCC and compared them with alterations of A antigen expression in TCC, dysplasia and normal urothelium. A total of 81 samples of TCC of the bladder obtained from transurethral resection ( TUR) ( n = 44) and radical cystectomy ( n = 37) were examined. Expression of the A antigen was evaluated by immunohistochemical staining ( IHC) using anti-A antigen monoclonal antibody. LOH of the ABO gene locus was examined by blunt-end single-strand DNA conformational polymorphism ( SSCP) analysis using flouresence-based auto sequencer. Promoter hypermethylation of the ABO gene were examined by bisulfite PCR-SSCP ( BiPS) analysis and/or methylation-specific PCR ( MSP). Loss of A allele and/or hypermethylation were significantly associated with abnormal expression of the A antigen in cases undergoing TUR ( P = 0.02) and radical cystectomy ( P = 0.0005). For the analysis of the concomitant dysplasia in 23 cases with TCC of the bladder, the expression of the A antigen was maintained, regardless of the A allelic loss or methylation status in the tumor. In conclusion, A allelic loss and hypermethylation in the promoter region of the ABO gene showed significant correlation with reduction of A antigen expression in TCC, while the expression of the A antigen is maintained in concomitant dysplasia or normal urothelium, suggesting that loss of the ABO gene and/or its promoter hypermethylation is a specific marker for TCC.

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Increased DNA methyltransferase 1 (DNMT1) protein expression in precancerous conditions and ductal carcinomas of the pancreas.

Peng DF, Kanai Y, Sawada M, Ushijima S, Hiraoka N, Kosuge T, Hirohashi S.

Cancer Sci (BLACKWELL PUBLISHING) 96 ( 7 ) 403 - 408 2005

Accepted, ISSN 13479032

Aberrant DNA methylation has been shown to play an important role during multistage carcinogenesis in various human organs. The aim of the present study was to evaluate the significance of DNA methyltransferase 1 (DNMT1) protein expression during pancreatic carcinogenesis. Immunohistochemical analysis of DNMT1 in 48 peripheral pancreatic duct epithelia showing no remarkable histological findings without an inflammatory background (DE), 54 peripheral pancreatic duct epithelia with an inflammatory background (DEI), 188 pancreatic intraepithelial neoplasias (PanIN), and 220 areas of invasive ductal carcinoma from surgical specimens resected from 100 patients, was carried out. The average incidence of DNMT1 immunoreactivity increased progressively from DE to DEI (P = 0.003), from DE and DEI to PanIN (P < 0.0001), among PanIN with different grades of dysplasia (from PanIN I to PanIN II, P = 0.0012), from PanIN to invasive ductal carcinomas (P < 0.0001) and among invasive ductal carcinomas with different grades of histological differentiation (from well or moderately to poorly differentiated adenocarcinomas, P < 0.0001). High-level DNMT1 protein expression in invasive ductal carcinomas was correlated significantly with an advanced t category (P=0.0224) and an advanced stage (P=0.0294). Moreover, patients with invasive ductal carcinomas showing high-level DNMT1 protein expression had a poorer outcome (P = 0.0469). These data suggest that increased DNMT1 protein expression participates in multistage pancreatic carcinogenesis from the precancerous stage to malignant progression of ductal carcinomas and may be a biological predictor of poor prognosis.

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DNA hypermethylation on multiple CpG islands associated with increased DNA methyltransferase DNMT1 protein expression during multistage urothelial carcinogenesis.

Nakagawa T, Kanai Y, Ushijima S, Kitamura T, Kakizoe T, Hirohashi S

J Urol (LIPPINCOTT WILLIAMS & WILKINS) 173 ( 5 ) 1767 - 1771 2005

Accepted, ISSN 00225347

Purpose: We elucidated the significance of aberrant DNA methylation on multiple CpG islands and its correlation with DNA methyltransferase DNMT1 protein expression during urothelial carcinogenesis.
Materials and Methods: We examined the DNA methylation status on multiple CpG islands by methylation specific polymerase chain reaction and combined bisulfite restriction enzyme analysis in 12 specimens of normal urothelium, 23 of noncancerous urothelium showing no remarkable histological changes obtained from patients with bladder cancer (NBC) and 70 of transitional cell carcinoma (TCC).
Results: DNA methylation on CpG islands of the p16 (0%, 17% and 21%) and death-associated protein kinase (13%, 33% and 29%) genes, and methylated in tumor-2 (56%, 60% and 76%), 12 (0%, 6% and 30%), 25 (25%, 27% and 35%) and 31 (45%, 56% and 79%) clones was detected in normal urothelium, NBCs and TCCs, respectively. The incidence of concurrent DNA hypermethylation on 3 or more CpG islands in NBCs (38%) was significantly higher than that in normal urothelium (0%, p = 0.0455) and even higher in TCCs (59%, p = 0.0043). The incidence of the CpG island methylator phenotype in nonpapillary carcinomas (nodular invasive carcinomas and their precursors, ie flat carcinoma in situ, 71%) was significantly higher than in papillary carcinomas (40%, p = 0.0143). In all specimens examined concurrent DNA hypermethylation on 3 or more CpG islands significantly correlated with immunohistochemically evaluated DNMT1 protein over expression (p = 0.0167).
Conclusions: DNA hypermethylation on multiple CpG islands in association with DNMT1 protein over expression may participate in multistage urothelial carcinogenesis even at the precancerous stage and particularly in the development of nodular invasive carcinomas of the bladder.

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

Nam J., Ino Y., Kanai Y., Sakamoto M., Hirohashi S.

Clinical and Experimental Metastasis (Clinical and Experimental Metastasis) 21 ( 1 ) 49 - 56 2004.03

ISSN 02620898

Down-regulation of the E-cadherin-mediated cell adhesion system is strongly related to cancer invasion and metastasis. Aberrant CpG hypermethylation in the promoter region of the E-cadherin gene has been shown to be responsible for reduction of E-cadherin expression. The present study was designed to test the hypothesis that the demethylating agent 5-aza-2′-deoxycytidine (AZA) can restore the E-cadherin system and reduce the potential for metastasis. AZA treatment modified the methylation status of the 5′ CpG island in the E-cadherin promoter, and induced re-expression of E-cadherin in human cancer cells whose E-cadherin expression had been silenced. The re-expressed E-cadherin was correlated with increased in vitro aggregation and reduced motility. After inoculation of cancer cells (MDA-MB-435S) into the mammary fat pads of mice with severe combined immunodeficiency, the mice were treated for nine consecutive weeks with AZA three times per week i.p. The AZA treatment suppressed both growth of the primary tumor and lung metastasis in comparison with untreated controls, suggesting that the suppression of metastasis may be, at least partly, attributable to restoration of E-cadherin expression. Therefore, inhibition of DNA methylation may be useful for preventing cancer metastasis.

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Alterations in gene expression associated with the overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, during human hepatocarcinogenesis.

Kanai Y, Saito Y, Ushijima S, Hirohashi S.

J Cancer Res Clin Oncol (SPRINGER) 130 ( 11 ) 636 - 644 2004

Accepted, ISSN 01715216

Purpose: Overexpression of a splice variant of DNA methyltransferase 3b, DNMT3b4, correlates significantly with DNA hypomethylation in pericentromeric satellite regions, which is known to result in centromeric decondensation and enhanced chromosomal recombination in precancerous conditions and hepatocellular carcinomas (HCCs). We aimed to elucidate further the significance of DNMT3b4 during human hepatocarcinogenesis. Methods: DNMT3b4-transfected human epithelial 293 cells were characterized using growth rate measurements, gene expression microarray, and quantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses. RT-PCR was also performed on eight normal liver specimens, 45 noncancerous liver specimens showing chronic hepatitis or cirrhosis, which are considered to be precancerous conditions, and 56 HCCs. Results: The growth rate of the DNMT3b4 transfectants was about double that of mock-transfectants. Induction of signal transducer and activator of transcription 1 (STAT1), an effector of interferon signaling, and of a set of downstream genes implicated in such signaling, was observed in the DNMT3b4 transfectants. There was significant correlation between the mRNA expression levels of DNMT3b4 and STAT1 in HCCs. mRNA expression levels of STAT1 and the three downstream genes examined were all significantly elevated in the chronic hepatitis and cirrhosis specimens compared with the normal liver specimens. Among the HCCs, the mRNA expression levels of STAT1 and the downstream genes were higher in tumors without portal vein involvement than in more malignant HCCs with portal vein involvement. Significant correlations between the mRNA expression levels of STAT1 and each of the downstream genes were observed in the tissue samples. Conclusions: Overexpression of DNMT3b4 is involved in human hepatocarcinogenesis, even at the precancerous stages, not only by inducing chromosomal instability but also by affecting the expression of specific genes.

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Collecting duct carcinoma of the kidney presenting uncommon findings.

Maeda T, Tateishi U, Nakagawa T, Ojima H, Kanai Y, Fujimoto H.

Eur J Radiol Extra (Elsevier BV) 49 ( 1 ) 25 - 29 2004

Accepted, ISSN 1571-4675

We present a case of collecting duct carcinoma (CDC) of the kidney exhibiting uncommon radiologie findings. A 67-year-old man had a history of hepatic resection for hepatocellular carcinomas. Follow-up CT revealed a left renal mass that presented an exophytic appearance with distortion of the renal contour. Since malignancy was suspected, the patient underwent a left radical nephrectomy with partial adrenalectomy and para-aortic lymph nodes dissection. Histologically, the majority of the tumor showed a tubular structure, and the rest was a solid area resembling transitional cell carcinoma (TCC). A diagnosis of CDC was made. In previous reports, common radiologie findings of CDC have been a medullary location and infiltrative appearance. Therefore, the outer contour of the affected kidney is usually maintained when the mass is relatively small. However, the present case did not show these common findings. In conclusion, the radiologic findings of CDC may be various and we should consider this tumor in the differential diagnosis of renal tumors. © 2003 Elsevier Ireland Ltd. All rights reserved.

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Prognostic significance of dysadherin expression in tongue cancer: immunohistochemical analysis of 91 cases.

Nakanishi Y, Akimoto S, Sato Y, Kanai Y, Sakamoto M, Hirohashi S.

Appl Immunohistochem Mol Morphol (LIPPINCOTT WILLIAMS & WILKINS) 12 ( 4 ) 323 - 328 2004

Accepted, ISSN 15412016

The E-cadherin-mediated cell adhesion system is frequently inactivated by multiple mechanisms and is involved in tumor progression in many types of cancer. Recently we have reported a novel cell membrane glycoprotein, dysadherin, which has an anti-cell-cell adhesion function and downregulates E-cadherin. Expressions of dysadherin and E-cadherin were investigated immunohistochemically in 91 patients with squamous cell carcinoma of the tongue to determine the correlation between the 2 molecules and their associations with the clinicopathologic features of the tumors and with patient survival. Dysadherin was expressed at the cell membranes of many cancer cells. Twenty-five percent of the tumors showed dysadherin immunopositivity in more than 50% of the cancer cells. Sixty-nine percent of the tumors showed reduced E-cadherin immunopositivity. There was an inverse correlation between dysadherin expression and E-cadherin expression (P = 0.0001). Increased dysadherin expression was significantly correlated with an infiltrative type of growth pattern (P = 0.001), high tumor-node-metastasis (TNM) stage (P= 0.024), and poor patient survival (P = 0.003). After adjusting for growth pattern, TNM stage, and other clinicopathologic features, increased dysadherin expression and reduced E-cadherin expression were both significant predictors of poor survival (P = 0.0006). Increased dysadherin expression is a significant indicator of poor prognosis in patients with squamous cell carcinoma of the tongue.

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Laminin 5 β3 and γ2 chains are frequently coexpressed in cancer cells.

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

Pathol Int (BLACKWELL PUBLISHING ASIA) 54 ( 9 ) 688 - 692 2004

Accepted, ISSN 13205463

Laminin 5 plays an important role in cell migration during tumor invasion and tissue remodeling. However, previous studies have not clarified whether laminin 5 beta3 chain is coexpressed with laminin 5 gamma 2 chain in cancer cells. The present immunohistochemical study investigated the distribution of the laminin 5 beta3 and gamma 2 chains in 20 cases of squamous cell carcinoma of the tongue and 17 cases of colorectal carcinoma. Laminin 5 beta3 and gamma 2 chains were expressed in the cytoplasm of tumor cells. Tumor cells at the cancer-stromal interface and at the invasive front frequently showed immunopositivity for laminin 5 beta3 and gamma 2 chains in both squamous cell carcinoma of the tongue and colorectal carcinoma. Furthermore, strong expressions of these two proteins were observed in cancer cells invading in a scattered manner. Laminin 5 beta3 expression correlated significantly with laminin 5 gamma 2 expression in 20 cases of squamous cell carcinoma of the tongue (P=0.0002) and 17 cases of colorectal carcinoma (P<0.0001). These results suggest that in squamous cell carcinoma of the tongue and colorectal carcinoma, laminin 5 gamma 2 chain and beta3 chain are both important in the invasiveness of cancer cells.

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Expression of enamel proteins and LEF1 in adamantinomatous craniopharyngioma: evidence for its odontogenic epithelial differentiation.

Sekine S, Takata T, Shibata T, Mori M, Morishita Y, Noguchi M, Uchida T, Kanai Y, Hirohashi, S.

Histopathology (BLACKWELL PUBLISHING LTD) 45 ( 6 ) 573 - 579 2004

Accepted, ISSN 03090167

Aims: Adamantinomatous craniopharyngioma (ACP) resembles histologically some odontogenic tumours, such as ameloblastoma and calcifying odontogenic cyst. However, there has been no evidence that ACP differentiates also functionally as odontogenic epithelium. The aim of this study was to gain evidence of odontogenic epithelial differentiation in ACP by means of immunohistochemistry. Among normal human tissues, enamel proteins are expressed exclusively in teeth, and lymphoid enhancer factor 1 (LEF1), in co-operation with beta-catenin, play an important role in tooth development. The expression of these proteins is therefore indicative of odontogenic epithelial differentiation.
Methods and results: The expression of enamel proteins and LEF1 was examined in 10 adamantinomatous and six papillary craniopharyngiomas. All the ACPs showed a variable degree of enamel protein expression, including amelogenin, enamelin and enamelysin, mainly in ghost cells. LEF1 was also heterogeneously expressed in ACPs; remarkably, its expression pattern was identical to that of nuclear beta-catenin accumulation. In contrast, none of the papillary craniopharyngiomas expressed enamel proteins or LEF1.
Conclusions: These results suggest that ACP consistently shows odontogenic epithelial differentiation. Since ACPs harbour beta-catenin mutation, the inappropriate activation of beta-catenin/LEF1 complex-dependent transcription may play a critical role in ACP tumorigenesis.

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Cytoplasmic p120ctn regulates the invasive phenotypes of E-cadherin-deficient breast cancer.

Shibata T, Kokubu A, Sekine S, Kanai Y, Hirohashi S.

Am J Pathol (AMER SOC INVESTIGATIVE PATHOLOGY, INC) 164 ( 6 ) 2269 - 2278 2004

Accepted, ISSN 00029440

In a search for signalling molecules that act downstream of E-cadherin inactivation in cancer, we examined the expression and localization of E-cadherin-associated proteins in lobular carcinoma, in which the E-cadherin gene is frequently inactivated, and found that E-cadherin down-regulation correlated with the cytoplasmic localization of p120ctn. Similar cytoplasmic localization of p120ctn and growth factor-induced accumulation of tyrosine-phosphorylated p120ctn in the protrusive domain were observed in E-cadherin-deficient breast cancer cells. Down-regulation of endogenous p120ctn by RNA interference promoted stress fiber formation and induced a flattened morphology with an increase of Rho-GTPase activity; it also reduced the development of membranous protrusions and migratory activity in E-cadherin-deficient breast cancer cells. Inactivation of E-cadherin in cancer cells is associated with the conversion from epithelial to mesenchymal phenotype, which also occurs in physiological conditions such as developmental processes. Cytoplasmic localization of p120ctn accompanied by E-cadherin down-regulation was observed in mesoderm cells that had undergone epithelial-mesenchymal transition during early mouse embryogenesis. Collectively, our results suggest that cytoplasmic p120ctn may contribute to the invasive phenotype of E-cadherin-deficient breast cancer cells.

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

Shimamura T, Yasuda J, Ino Y, Gotoh M, Tsuchiya A, Nakajima A, Sakamoto M, Kanai Y, Hirohashi S.

Cancer Res (AMER ASSOC CANCER RESEARCH) 64 ( 19 ) 6989 - 6995 2004

ISSN 00085472

Dysadherin is a membrane glycoprotein expressed strongly in several human cancers; Overexpression of dysadherin in tumor cells is closely associated with malignant phenotype (e.g., metastasis) and poor prognosis. In our analysis,,six pancreatic cancer cell lines showed a positive correlation between dysadherin expression and cell motility. Introduction of small interfering RNA (siRNA) against dysadherin into the Panc-1 cell line caused reduction of dysadherin expression and suppression of cell motility. In contrast, stable transfection of a dysadherin expression vector into the Capan-1 cell line increased cell motility. In vivo, the metastatic potential of orthotopically transplanted Capan-1 tumor cells in severe combined immunodeficient mice was increased by dysadherin overexpression. Cell morphology and actin organization were also influenced by modulation of dysadherin expression. Cells transfected with dysadherin siRNA tended to have a relatively larger, more spread shape and increased transverse actin stress fibers compared with parent cells and cells transfected with control siRNA. Our study suggests that dysadherin is able to modulate actin structures, stimulate cell motility, and contribute directly to the metastatic potential of human pancreatic cancer cells.

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5-aza-2'-deoxycytidine restores the E-cadherin system in E-cadherin-silenced cancer cells and reduces cancer metastasis.

Nam JS, Ino Y, Kanai Y, Sakamoto M, Hirohashi S.

Clin Exp Metastasis (KLUWER ACADEMIC PUBL) 21 ( 1 ) 49 - 56 2004

Accepted, ISSN 0262-0898

Down-regulation of the E-cadherin-mediated cell adhesion system is strongly related to cancer invasion and metastasis. Aberrant CpG hypermethylation in the promoter region of the E-cadherin gene has been shown to be responsible for reduction of E-cadherin expression. The present study was designed to test the hypothesis that the demethylating agent 5-aza-2'-deoxycytidine (AZA) can restore the E-cadherin system and reduce the potential for metastasis. AZA treatment modified the methylation status of the 5' CpG island in the E-cadherin promoter, and induced re-expression of E-cadherin in human cancer cells whose E-cadherin expression had been silenced. The re-expressed E-cadherin was correlated with increased in vitro aggregation and reduced motility. After inoculation of cancer cells (MDA-MB-435S) into the mammary fat pads of mice with severe combined immunodeficiency, the mice were treated for nine consecutive weeks with AZA three times per week i.p. The AZA treatment suppressed both growth of the primary tumor and lung metastasis in comparison with untreated controls, suggesting that the suppression of metastasis may be, at least partly, attributable to restoration of E-cadherin expression. Therefore, inhibition of DNA methylation may be useful for preventing cancer metastasis.

Malignant mixed epithelial and stromal tumours of the kidney: a report of the first two cases with a fatal clinical outcome.

Nakagawa T, Kanai Y, Fujimoto H, Kitamura H, Furukawa H, Maeda S, Oyama T, Takesaki T, Hasegawa T.

Histopathology (BLACKWELL PUBLISHING LTD) 44 ( 3 ) 302 - 304 2004

ISSN 0309-0167

Increased DNA methyltransferase 1 (DNMT1) protein expression correlates significantly with poorer tumor differentiation and frequent DNA hypermethylation of multiple CpG islands in gastric cancers.

Etoh T, Kanai Y, Ushijima S, Nakagawa T, Nakanishi Y, Sasako M, Kitano S, Hirohashi S.

Am J Pathol (AMER SOC INVESTIGATIVE PATHOLOGY, INC) 164 ( 2 ) 689 - 699 2004

Accepted, ISSN 00029440

We evaluated the significance of aberrant DNA methyltransferase I (DNMT1) protein expression during gastric carcinogenesis. The protein expression of DNMT1, Muc2, human gastric mucin, E-cadherin, and proliferating cell nuclear antigen was examined immunohistochemically in gastric cancers and corresponding noncancerous mucosae from 134 patients. The DNA methylation status of the CpG islands of the p16, human MutL homologue I (hMLH1), E-cadherin, and thrombospondin-1 (THBS-1) genes and the methylated in tumor (MINT)-1, -2, -12, and -31 clones was examined by methylation-specific polymerase chain reaction and combined bisulfite restriction enzyme analysis. Epstein-Barr virus (EBV) infection was detected by in situ hybridization. Nuclear immunoreactivity for DNMT1 was not detected in any of the noncancerous epithelia, except in proliferative zones (positive internal control), but was found in 97 (72%) of the gastric cancers. DNMT1 overexpression correlated significantly with poorer tumor differentiation (P < 0.001), but not with the phenotype (gastric type versus intestinal type) of the cancer cells. it also correlated significantly with DNA hypermethylation of the CpG islands of the hMLH1 (P = 0.024) and THBS-1 genes (P = 0.043), and with the CpG island methylator phenotype in the gastric cancers (P = 0.007). Reduced E-cadherin expression correlated significantly with poorer tumor differentiation (P = 0.002), DNA hypermethylation of the E-cadherin gene (P < 0.001) and DNMT1 overexpression (P = 0.014). DNMT1 overexpression was also associated with EBV infection (a potential etiological factor in gastric carcinogenesis) but not with the proliferative activity of the cancer cells as indicated by the proliferating cell nuclear antigen-labeling index. These results suggest that DNMT1 overexpression may not be just a secondary effect of increased cancer cell proliferative activity, but may be associated with EBV infection and other etiological factors during gastric carcinogenesis. Furthermore, DNMT1 may play a significant role in the development of poorly differentiated gastric cancers by inducing frequent DNA hypermethylation of multiple CpG islands.

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Downregulation and growth inhibitory effect of epithelial-type Krüppel-like transcription factor KLF4, but not KLF5, in bladder cancer

Ohnishi S., Ohnami S., Laub F., Aoki K., Suzuki K., Kanai Y., Haga K., Asaka M., Ramirez F., Yoshida T.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications) 308 ( 2 ) 251 - 256 2003.08

ISSN 0006291X

Krüppel-like factors (KLFs) are key transcriptional regulators of cell differentiation and proliferation. Among the KLF family, the expression of KLF4 (GKLF) and KLF5 (IKLF) is highly restricted in the epithelial cells of several organs such as the gut and skin, and it has been reported that these epithelial-type KLF genes may be involved in colon carcinogenesis. Recently we found that Klf4 and Klf5 genes were significantly expressed in the developmental bladder epithelium of mice as well. Therefore, in this report we studied the involvement of the KLF4 and KLF5 genes in bladder carcinogenesis. First, we analyzed the expression of KLF4 and KLF5 in a variety of human bladder cancer cell lines and surgical specimens by RNA blot and in situ hybridization analyses. Both genes were highly expressed in the normal bladder epithelium, whereas KLF4, but not KLF5, was frequently downregulated in bladder cancer cell lines and cancer tissues. We then transduced the KLF4 and KLF5 genes into the bladder cancer cell lines using adenoviral vectors to examine the biological activities of the genes on those cells. The transduction of KLF4, but not KLF5, suppressed cell growth and induced apoptosis. Our study suggests that inactivation of KLF4 is one of the frequent steps towards bladder carcinogenesis. © 2003 Elsevier Inc. All rights reserved.

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Cell adhesion system and human cancer morphogenesis.

Hirohashi S. Kanai Y.

Cancer Sci (JAPANESE CANCER ASSOC) 94 ( 7 ) 575 - 581 2003

Accepted, ISSN 00720151

Cell-cell adhesion determines the polarity of cells and participates in the maintenance of the cell societies called tissues. Cell-cell adhesiveness is generally reduced in human cancers. Reduced intercellular adhesiveness allows cancer cells to disobey the social order, resulting in destruction of histological structure, which is the morphological hallmark of malignant tumors. Reduced intercellular adhesiveness is also indispensable for cancer invasion and metastasis. A tumor-suppressor gene product, E-cadherin, and its undercoat proteins, catenins, which connect cadherins to actin filaments, are located at lateral borders, concentrating on adherens junctions, of epithelial cells and establish firm cell-cell adhesion. The E-cadherin cell adhesion system in cancer cells is inactivated by various mechanisms that reflect the morphological and biological characteristics of the tumor. Silencing of the E-cadherin gene by DNA hypermethylation around the promoter region occurs frequently, even in precancerous conditions. In diffuse infiltrating cancers, mutations are found in the genes for E-cadherin and alpha- and beta-catenins. At the invading front of cancers, the E-cadherin cell adhesion system is inactivated by tyrosine phosphorylation of beta-catenin; an oncogene product, c-erbB-2 protein, is found to associate directly with beta-catenin. The E-cadherin cell adhesion system cross-talks with the Wingless/Wnt signaling pathway through beta-catenin, and expression of genes, which participate in cancer morphogenesis, may be regulated in conjunction with the Wingless/Wnt signaling pathway. Dysadherin, a newly identified cancer-associated cell membrane glycoprotein, down-regulates E-cadherin and promotes cancer metastasis. In conclusion, inactivation of the E-cadherin cell adhesion system by both genetic and epigenetic mechanisms plays a significant role during multistage human carcinogenesis.

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Mutation of the DNA methyltransferase (DNMT) 1 gene in human colorectal cancers.

Kanai Y, Ushijima S, Nakanishi Y, Sakamoto M, Hirohashi S.

Cancer Lett (ELSEVIER SCI IRELAND LTD) 192 ( 1 ) 75 - 82 2003

Accepted, ISSN 03043835

Alteration of DNA methylation is one of the most consistent epigenctic changes in human cancers. DNA methyltransferase (DNMT) 1 is a major enzyme that determines genomic methylation patterns. In order to understand the significance of mutations of the DNMT1 gene during human carcinogenesis, we performed polymerase chain reaction-single strand conformation polymorphism analysis using 46 oligonucleotide primer sets for all 40 coding exons and the 5-flanking region (450 bp) of the DNMT1 gene in 29 colorectal cancers, 32 stomach cancers, 40 hepatocellular carcinomas (HCCs) and a corresponding sample of non-cancerous tissue from each case. Mutations in coding exons of the DNMT1 gene were detected in two (7%) of the colorectal cancers: they consisted of one-base deletion resulting in deletion of the whole catalytic domain and a point mutation resulting in a single amino acid substitution. No stomach cancers or HCCs showed mutations in the coding exons of the DNMT1 gene. No mutation in the 5'-flanking region of the DNMT1 gene was detected in any of the colorectal and stomach cancers or HCCs. These data suggest that mutational inactivation of the DNMT1 gene that potentially causes a genome-wide alteration of DNA methylation status may be a rare event during human carcinogenesis. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

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Increased protein expression of DNA methyltransferase (DNMT) 1 is significantly correlated with the malignant potential and poor prognosis of human hepatocellular carcinomas.

Saito Y, Kanai Y, Nakagawa T, Sakamoto M, Saito H, Ishii H, Hirohashi S.

Int J Cancer (WILEY-LISS) 105 ( 4 ) 527 - 532 2003

Accepted, ISSN 00207136

Alteration of DNA methylation is one of the most consistent epigenetic changes in human cancers. DNA methyltransferase (DNMT) 1 is a major enzyme involved in establishing genomic methylation patterns. Most of the studies concerning DNMT1 expression in human cancers have been performed only at the mRNA level. To directly examine DNMT1 protein expression levels during human hepatocarcinogenesis, 16 histologically normal liver tissues, 51 noncancerous liver tissues exhibiting chronic hepatitis or cirrhosis, which are considered to be precancerous conditions, and 53 hepatocellular carcinomas (HCCs) were subjected to immunohistochemic examination. If more than 20% of the cells exhibited nuclear DNMT1 staining, the tissue sample was considered to be DNMT1-positive. DNMT1 immunoreactivity was observed in 23 (43%) of the HCCs, but in none (0%) of the histologically normal liver or noncancerous liver tissues exhibiting chronic hepatitis or cirrhosis. The incidence of increased DNMT1 protein expression in HCCs correlated significantly with poor tumor differentiation (p = 0.0006) and portal vein involvement (p = 0.0002). Moreover, the recurrence-free (p = 0.0001) and overall (p < 0.0001) survival rates of patients with HCCs exhibiting increased DNMT1 protein expression were significantly lower than those of patients with HCCs that did not exhibit increased expression. Increased DNMT1 protein expression may play a critical role in the malignant progression of HCCs and be a biologic predictor of both HCC recurrence and a poor prognosis in HCC patients. (C) 2003 Wiley-Liss, Inc.

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Downregulation and growth inhibitory effect of epithelial-type Kruppel-like transcription factor KLF4, but not KLF5, in bladder cancer.

Ohnishi S, Ohnami S, Laub F, Aoki K, Suzuki K, Kanai Y, Haga K, Asaka M, Ramirez F, Yoshida T.

Biochem Biophys Res Commun 308 251 - 256 2003

Accepted

β-catenin mutations are frequent in calcifying odontogenic cysts, but rare in ameloblastomas.

Sekine S, Sato S, Takata T, Fukuda Y, Ishida T, Kishino M, Shibata T, Kanai Y, Hirohashi S.

Am J Pathol (AMER SOC INVESTIGATIVE PATHOLOGY, INC) 163 ( 5 ) 1707 - 1712 2003

Accepted, ISSN 00029440

We have reported previously that alterations to beta-catenin occur frequently in adamantinomatous craniopharyngioma. Based on its histological resemblance to some odontogenic tumors, we suspected the presence of common genetic alterations among these tumors. To address this issue, 11 cases of calcifying odontogenic cyst (COC) and 20 cases of ameloblastoma were investigated for the presence of beta-catenin mutations and beta-catenin expression. Ten COCs were successfully analyzed by direct sequencing, and nine of them were found to harbor somatic beta-catenin mutations. Immunohistochemically, all of the COCs showed nuclear and cytoplasmic expression of beta-catenin with a heterogeneous pattern. No beta-catenin mutations were found in ameloblastomas, except for one case of the follicular type. All follicular ameloblastomas exhibited moderate nuclear and cytoplasmic accumulation of beta-catenin, in contrast to the predominantly membranous expression seen in the plexiform type. beta-Catenin mutation is considered to be a characteristic genetic feature of COC, and may play a critical role in its histogenesis. Although ameloblastoma closely resembles COC histologically, the two have genetically distinctive features.

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Overexpression of KIT in chromophobe renal cell carcinoma.

Yamazaki K, Sakamoto M, Ohta T, Kanai Y, Ohki M, Hirohashi S.

Oncogene (NATURE PUBLISHING GROUP) 22 ( 6 ) 847 - 852 2003

Accepted, ISSN 09509232

We analysed gene-expression profiles in 15 surgical specimens of conventional, papillary, and chromophobe renal cell carcinomas (RCCs) using high-density oligonucleotide arrays. From about 12 000 genes targeted by the array, 67 were upregulated specifically in each histological type of RCC. The oncogene KIT was one of the genes whose expression was upregulated specifically in chromophobe RCCs. Immunohistochemical analysis demonstrated the KIT gene product on the cell membrane of chromophobe RCC in all cases, although it was not detected in conventional RCCs or non-neoplastic kidneys except for weak staining in the cytoplasm of renal tubules. These results suggest that each histological subtype of RCC has a unique gene-expression profile, and in particular indicates for the first time that KIT could be a useful marker for chromophobe RCC. As overexpression of KIT might be involved in tumor growth, KIT could be a new therapeutic target in this special type of RCC.

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Hypermethylation of an E-cadherin (CDH1) promoter region in high grade transitional cell carcinoma of the bladder comprising carcinoma in situ.

Horikawa Y, Sugano K, Shigyo M, Yamamoto H, Nakazono M, Fujimoto H, Kanai Y, Hirohashi S, Kakizoe T, Habuchi T, Kato T.

J Urol (LIPPINCOTT WILLIAMS & WILKINS) 169 ( 4 ) 1541 - 1545 2003

Accepted, ISSN 00225347

Purpose: We elucidated the role of methylation in the promoter region of the CDH1 gene in bladder carcinogenesis, particularly in those comprising carcinoma in situ.
Materials and Methods: A total of 49 cases of transitional cell carcinoma of the bladder obtained from transurethral resection were examined. Methylation status of the CDH1 promoter region was analyzed by methylation specific polymerase chain reaction from chemically modified DNA after Na-bisulfite treatment. Loss of heterozygosity on 16q was examined by blunt end single strand DNA conformation polymorphism using 4 tetranucleotide repeat microsatellite markers assigned on 16q13 to 22.1. E-cadherin expression was evaluated by immunostaining on formalin fixed, paraffin embedded tissue sections using anti E-cadherin murine monoclonal antibody, HECD1 and standard avidin-biotin immunoperoxidase complex technique.
Results: Analysis of the 49 bladder transitional cell carcinoma samples showed CDH1 promoter methylation in 23 (47%). Methylation of the CDH1 gene did not correlate with tumor stage (p = 0.2097) but with high grade transitional cell carcinoma (p = 0.0416). CDH1 promoter methylation was observed at a significantly higher frequency in the carcinoma in situ positive group than in the carcinoma in situ negative group (16 of 18 cases or 89% versus 7 of 31 or 23%, p <0.0001) and it strongly correlated with abnormal E-cadherin expression (p <0.0001). We found 16q loss of heterozygosity in 16 of 47 cases (34%), which correlated with higher histological grade (p = 0.0069) but not with the presence of the carcinoma in situ component (p = 0.1235).
Conclusions: This study showed that CDH1 gene promoter methylation is strongly associated with bladder transitional cell carcinoma comprising carcinoma in situ.

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Increased DNA methyltransferase 1 protein expression in human transitional cell carcinoma of the bladder.

Nakagawa T, Kanai Y, Saito Y, Kitamura T, Kakizoe T, Hirohashi S.

J Urol (LIPPINCOTT WILLIAMS & WILKINS) 170 ( 6 I ) 2463 - 2466 2003

Accepted, ISSN 00225347

Purpose: DNA methylation is a key regulator of gene transcription and genomic stability, and alteration of DNA methylation is one of the most consistent epigenetic changes in human cancers. We elucidated the significance of aberrant protein expression of DNA methyltransferase (DNMT) 1, a major enzyme involved in the determination of genomic methylation patterns, during human urothelial carcinogenesis.
Materials and Methods: A total of 61 samples of normal urothelium, 89 noncancerous urothelium. samples showing no remarkable histological changes from patients with bladder cancer (NBCs), 78 dysplastic urothelium samples and 174 transitional cell carcinoma samples (TCCs) were subjected to immunohistochemical analysis for DNMT1.
Results: The incidence of nuclear DNMT1 immunoreactivity in NBCs (65%) was significantly higher than in normal urothelium (20%, p <0.0001) and the incidence was even higher in dysplastic urothelium samples (84%, p = 0.0017). The incidence of nuclear DNMT1 immunoreactivity was 87% in TCCs and the intensity of nuclear immunoreactivity was markedly increased in TCCs compared with that in dysplastic urothelium samples. DNMT1 expression levels had already increased in NBCs in which the proliferating cell nuclear antigen labeling index had not yet increased. Increased DNMT1 protein expression correlated significantly with histological grade (p <0.0001). DNMT1 protein expression was higher in nonpapillary tumors (p = 0.0001), especially flat carcinoma in situ, than in papillary tumors.
Conclusions: Progressively increasing expression of DNMT1 protein is not entirely a secondary result of increased cell proliferative activity, but rather it is associated with urothelial carcinogenesis even during the precancerous stages. In particular, it is associated with the development of flat carcinoma in situ, which is considered to be a precursor of nodular invasive carcinoma of the bladder.

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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, Hirohashi S.

Proc Natl Acad Sci USA (Proceedings of the National Academy of Sciences of the United States of America) 99 ( 15 ) 10060 - 10065 2002

Accepted, ISSN 00278424

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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Pancreatic metastasis from renal cell carcinoma extending into the main pancreatic duct: a case report.

Yachida S, Fukushima N, Kanai Y, Nimura S, Shimada K, Yamamoto J, Sakamoto M.

Jpn J Clin Oncol (OXFORD UNIV PRESS) 32 ( 8 ) 315 - 317 2002

Accepted, ISSN 03682811

While metastasis to the pancreas is uncommon, it may occur from renal cell carcinomas (RCCs). We here present a case of pancreatic metastasis from RCC extending into the main pancreatic duct (MPD) in a 66-year-old Japanese man. The patient had a history of RCC treated with a radical nephrectomy 17 years previously and was found to have a mass similar to2 cm in diameter in the body of the pancreas on radiological images. The patient was suspected of having pancreatic metastasis from RCC and underwent a distal pancreatectomy with splenectomy. Histologically, the tumor consisted of cells arranged in trabecular and alveolar structures with clear or eosinophilic granular cytoplasm, compatible with a metastatic RCC. The pancreatic tumor extended into the MPD with the stream of pancreatic juice. This condition is similar to RCC extension into the renal vein and the inferior vena cava. In conclusion, although extension into the MPD may be rare, such a growth pattern may be characteristic of metastases from RCCs.

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Correlation of DNA hypomethylation at pericentromeric heterochromatin regions of chromosomes 16 and 1 with histological features and chromosomal abnormalities of human breast carcinomas.

Tsuda H, Takarabe T, Kanai Y, Fukutomi T, Hirohashi S.

Am J Pathol (AMER SOC INVESTIGATIVE PATHOLOGY, INC) 161 ( 3 ) 859 - 866 2002

Accepted, ISSN 00029440

Changes in DNA methylation status are not only important for regulating gene expression but are also suggested to induce chromosome instability. To reveal the correlation of DNA methylation status in heterochromatin regions with tumor histology and with chromosome alterations, DNA methylation status was examined by Southern blot analysis, and numerical and structural chromosome alterations, including the formation of der(16)t(1;16)/der(1;16), were examined by fluorescence in situ hybridization at the two loci in the pericentromeric satellite 2 regions of chromosomes 16 and 1 in 39 human breast carcinomas. DNA hypomethylation at the D16Z3 and the D1Z1 loci was detected in 31% (12 of 39) and 36% (12 of 33) of carcinomas, respectively, and mostly concurred. DNA hypomethylation was more frequent in the carcinoma group of more aggressive histological types or grade 3 than in the carcinoma of less aggressive histological types or grades 1 and 2, and tended to be more frequent in carcinomas with greater than or equal to4 copies of chromosomes 16 and/or 1 than in carcinomas with less than or equal to3 copies of any of these chromosomes. The frequency of DNA hypomethylation at the D16Z3 and the D1Z1 loci was 45% (10 of 22) and 53% (9 of 17) in carcinomas without der(16)t(1;16)/der(1;16), formation, but only 12% (2 of 17) and 19% (3 of 16) in carcinoma with der(16)t(1;16)/der(1;16), respectively (P = 0.036 and 0.070). The 16q breakage was almost equally detected between carcinoma groups with and without the DNA hypomethylation. DNA hypomethylation in the satellite 2 regions was suggested to be associated with the accumulation of a large number of numerical chromosome alterations and involved in the development of breast carcinomas of aggressive histological features. on the contrary, chromosome instability induced by mechanisms other than DNA hypomethylation in heterochromatin regions might cause the formation of der(16)t(1;16)/der(1;16) and less aggressive breast carcinomas.

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DNA methylation

Kanai Y

Nippon rinsho. Japanese journal of clinical medicine (Nippon rinsho. Japanese journal of clinical medicine) 59 Suppl 6 142 - 146 2001.10

ISSN 00471852

DNA methyltransferase expression and DNA methylation of CpG islands and peri-centromeric satellite regions in human colorectal and stomach cancers.

Kanai Y, Ushijima S, Kondo Y, Nakanishi Y, Hirohashi, S.

Int J Cancer (International Journal of Cancer) 91 ( 2 ) 205 - 212 2001

Accepted, ISSN 00207136

We evaluated the significance of aberrant DNA methyltransferase expression in human carcinogenesis by examining 32 colorectal and 34 stomach cancers. Levels of mRNAs encoding DNA methyltransferases were measured by reverse transcription, followed by real-time quantitative detection of PCR products. The DNA methylation state of CpG islands and peri-centromeric satellite regions was examined by bisulfite modification and Southern blotting, respectively. The average level of mRNA for DNMTI and DNMT3b in colorectal and stomach cancers was significantly higher than in corresponding non-cancerous mucosae, whereas the average level of mRNA for DNMT2 was significantly lower in colorectal and stomach cancers than in non-cancerous tissue. Over-expression of DNMT3b in stomach cancer was significantly higher in cases with lymph node metastasis than in cases without. DNA hypermethylation on the p16, human Mut L homologue-1 and thrombospondin-1 genes and the methylated in tumor (MINT) 1, 2, 12, 25 and 31 clones was found in 23%, 27%, 9%, 23%, 20%, 23%, 20% and 10% of the colon cancers and in 9%, 19%, 30%, 25%, 34%, 19%, 81% and 3% of the stomach cancers, respectively. Criteria for identification of the CpG island methylator phenotype (CIMP) were met in 23% of colorectal cancers and 31% of stomach cancers. DNA hypomethylation on satellites 2 and 3 was detected in 0% and 8% of colorectal and stomach cancers, respectively. Over-expression of DNMTI mRNA was significantly associated with CIMP, whereas the level of DNMT3b mRNA was not associated with CIMP or DNA hypomethylation of pericentromeric satellite regions. These data suggest that both over-expression of the maintenance DNA methyltransferase DNMTI and over-expression of a newly identified de novo DNA methyltransferase, DNMT3b, are involved in human carcinogenesis, probably at different stages and through different mechanisms. © 2001 Wiley-Liss, Inc.

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Intraductal papillary components in invasive ductal carcinoma of the pancreas are associated with long-term survival of patients.

Fukushima N, Sakamoto M, Mukai K, Kanai Y, Shimada K, Kosuge T, Hirohashi S.

Hum Pathol (W B SAUNDERS CO) 32 ( 8 ) 834 - 841 2001

Accepted, ISSN 00468177

Most patients with pancreatic ductal carcinoma have a poor prognosis. However, in certain cases, 5-year survival can be achieved after surgical resection. Analysis of the pathologic findings associated with good survival rates will assist in identifying the optimum treatment. The clinicopathologic features of 67 patients who underwent surgical resection of ductal adenocarcinoma of the pancreas between 1990 and 1996 were reviewed and correlated with survival rates. There were 42 men and 25 women, with a mean age of 62.1 years (range, 44 to 82 years). The mean greatest diameter of the tumor was 4.3 cm (range, 1.5 to 11 cm). Nineteen patients (29.4%) survived more than 3 years, and 9 (13.2%) survived more than 5 years after surgical resection. The intraductal papillary component (IDPC) of the carcinoma was the main focus of the pathologic observations. IDPC was defined as intraductal papillary proliferative lesions seen in the tumor nodule with proliferative cells consistent with carcinomatous cellular atypia. IDPC was clearly present (++) in 24 patients and vaguely present (+) in 9 patients. Using the Mantel-Cox test, a statistically significant correlation was found between the presence of IDPC (either + or ++) and postoperative patient survival (P = .002). IDPC is a morphologic feature associated with longer patient survival and should be taken into consideration in assessing the pathway of tumor progression. HUM PATHOL 32;834-841. (C) 2001 by W.B. Saunders Company.

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Expression of cadherins and their undercoat proteins (α-, β-, and γ-catenins and p120) and accumulation of β-catenin with no gene mutations in synovial sarcoma.

Sato H, Hasegawa T, Kanai Y, Tsutsumi Y, Osamura Y, Abe Y, Sakai H, Hirohashi S.

Virchows Arch (Virchows Archiv) 438 ( 1 ) 23 - 30 2001

Accepted, ISSN 09456317

E-cadherin, the major intercellular adhesion molecule of epithelial cells, is important in determining the architecture of sarcomas, especially those showing epithelioid features. In addition to its role in cell adhesion, β-catenin, a cadherin undercoat protein, has been shown to function as a downstream transcriptional activator of the Wnt/Wingless signaling pathway. In order to evaluate the significance of the cadherin cell adhesion system and the Wnt/Wingless signaling pathway in the morphogenesis and/or tumorigenesis of synovial sarcoma (a major type of sarcoma with epithelioid features), immunoreactivity for pan-cadherin, E-cadherin, and their undercoat proteins (α-, β-, and γ-catenins and p120) was evaluated in 15 synovial sarcomas. Immunoreactivity for pan-cadherin, E-cadherin, α-catenin, β-catenin, and p120 was observed in all 15 specimens. Immunoreactivity for pan-cadherin was stronger than that for E-cadherin. Expression of γ-catenin was detected in ten specimens. Although β-catenin was observed only at the cell-cell boundaries in four specimens, it was present in the nucleus and cytoplasm and at the cell-cell boundaries in the other 11, suggesting constitutional activation of the Wnt/Wingless signaling pathway in synovial sarcoma. Direct sequencing for exon 3 of the β-catenin gene, however, revealed no mutations in any of the 15 specimens. In conclusion, other types of cadherin besides E-cadherin, together with cadherin undercoat proteins, may play a role in cell adhesion in synovial sarcoma. Furthermore, mechanisms other than mutation of exon 3 of the β-catenin gene may activate the Wnt/Wingless signaling pathway in this type of tumor.

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

Hepatology (W B SAUNDERS CO) 33 ( 3 ) 561 - 568 2001

Accepted, ISSN 02709139

To evaluate the significance of alterations in DNA methylation during human hepatocarcinogenesis, we examined levels of mRNA for DNA methyltransferases and methyl-CpG-binding proteins and the DNA methylation status in 67 hepatocellular carcinomas (HCCs), The average level of mRNA for DNMT1 and DNMT3a was significantly higher in noncancerous liver tissues showing chronic hepatitis or cirrhosis than in histologically normal liver tissues, and was even higher in HCCs. Significant overexpression of DNMT3b and reduced expression of DNMT2 were observed in HCCs compared with the corresponding noncancerous liver tissues. DNA hypermethylation on CpG islands of the p16 (8% and 66%) and hMLH1 (0% and 0%) genes and methylated in tumor (MINT) 1 (6% and 34%), 2 (24% and 58%), 12 (21% and 33%), 25 (0% and 5%), and 31 (0% and 23%) clones, and DNA hypomethylation on satellites 2 and 3 (18% and 67%), were detected in noncancerous liver tissues and HCCs, respectively. There was no significant correlation between the expression level of any DNA methyltransferase and DNA methylation status. Reduced expression of DNA repair protein, MBD4, was significantly correlated with poorer tumor differentiation and involvement of portal vein. Slightly reduced expression of MBD2 was detected in HCCs, and the expression of MeCP2 was particularly reduced in HCCs with portal vein involvement. These data suggest that overexpression of DNMT1 and DNMT3a, DNA hypermethylation on CpG islands, and DNA hypomethylation on pericentromeric satellite regions are early events during hepatocarcinogenesis, and that reduced expression of MBD4 may play a role in malignant progression of HCC.

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mRNA expression of genes altered by 5-azacytidine treatment in cancer cell lines is associated with clinicopathological parameters of human cancers.

Kanai Y, Ushijima S, Saito Y, Nakanishi Y, Sakamoto M, Hirohashi S.

J Cancer Res Clin Oncol (SPRINGER-VERLAG) 127 ( 12 ) 697 - 706 2001

Accepted, ISSN 01715216

Objective: We attempted to identify tumor-related genes whose expression is affected by DNA methylation and that participate in the determination of the biological characteristics of human cancers. Methods and materials: We used the differential display method in ten 5-azacytidine-treated human cancer cell lines. Time-dependent induction of mRNA expression of Wip1. TROP2, B4-2 protein, BRCA-2 region transcription unit CG005, cofilin, ROCK, CD44, and interferon-inducible protein 6-16 was observed during 5-azacytidine treatment. We then evaluated the mRNA expression of these genes in 26 stomach and 32 colorectal cancers and 44 hepatocellular carcinomas (HCCs) and in the corresponding non-cancerous tissues. Results Significantly reduced rn RNA expression of Wip1, B4-2, and cofilin in stomach cancers, of Wip1, B4-2, BRCA-2 region transcription unit CG005, cofilin, and ROCK in colorectal cancers, and of TROP2, B4-2, and BRCA-2 region transcription unit CG005 in HCCs, was observed in comparison with the corresponding non-cancerous tissues, whereas overexpression of TROP2 was detected in colorectal cancers. Reduced expression of Wip1 and BRCA-2 region transcription unit CG005 was significantly correlated with poorer differentiation of HCCs, and mRNA expression of these genes was significantly reduced in HCCs associated with portal vein involvement compared with HCCs without such involvement. Expression of TROP2 mRNA was significantly higher in chronically diseased liver from HCC patients compared with histologically normal liver, but was reduced in progressed HCCs. Reduced expression of B4-2 protein was observed in association with hepatitis B virus infection of HCC patients. Conclusions: These data suggest that DNA methylation may play a role in human multistage carcinogenesis through direct or indirect regulation of multiple tumor-related genes.

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Aberrant DNA methylation precedes loss of heterozygosity on chromosome 16 in chronic hepatitis and liver cirrhosis.

Kanai Y, Ushijima S, Tsuda H, Sakamoto M, Hirohashi S.

Cancer Lett (Cancer Letters) 148 ( 1 ) 73 - 80 2000

Accepted, ISSN 03043835

The aim of this study was to examine the significance of aberrant DNA methylation, the participation of which in genetic instability is controversial, in hepatocarcinogenesis. The DNA methylation status of the region around the promoter of the E-cadherin tumor suppresser gene, which is located on 16q22.1, and the allelic status at the D16S421 locus, which is adjacent to the E-cadherin locus, were examined using microdissected liver specimens from 38 hepatocellular carcinoma (HCC) patients. Almost all of the non-cancerous liver tissues showed histological findings compatible with chronic hepatitis and cirrhosis, which are considered to be precancerous conditions. DNA hypermethylation was detected in 61% of the non-cancerous liver tissues. The incidence of DNA hypermethylation in the non-cancerous liver tissues of patients with HCCs also showing DNA hypermethylation (72%) was significantly higher than that of patients without DNA hypermethylation in their HCCs (53%, P<0.05). Loss of heterozygosity (LOH) at the D16S421 locus was detected in 35% of the non-cancerous liver tissues. The incidence of LOH in the non-cancerous liver tissues of patients with HCCs also showing LOH was 78%, whereas LOH was not detected in non-cancerous liver tissues of patients without LOH in their HCCs. Fifty-two percent of the non-cancerous liver tissues showed both or neither of DNA hypermethylation and LOH; the incidence of DNA hypermethylation alone in non-cancerous liver tissue was 41%. The incidence of LOH alone in non-cancerous liver tissue (7%) was significantly lower compared to those of the former two cases (P<0.0001). These data suggest that aberrant DNA methylation participates in the precancerous stage of hepatocarcinogenesis by preceding, or causing, LOH. Copyright (C) 2000 Elsevier Science Ireland Ltd.

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GSTP1 CpG island DNA hypermethylation in hepatocellular carcinomas.

Tchou JC, Lin X, Freije D, Isaacs WB, Brooks JD, Rashid A, De Marzo AM, Kanai Y, Hirohashi S, Nelson WG.

Int J Oncol 16 663 - 676 2000

Accepted

Signet-ring cell carcinoma of the urinary bladder associated with transitional cell carcinoma of the right ureter.

Kume H, Kanai Y, Tobisu K, Fujimoto H, Tomita K, Kakizoe T.

Scand J Urol Nephrol 34 278 - 279 2000

Accepted

Multistep carcinogenesis and DNA methylation.

Hirohashi S, Kanai Y.

Princess Takamatsu Symp 31 5 - 10 2000

Aberrant expression of β-catenin and mutation of exon 3 of the β-catenin gene in renal and urothelial carcinomas.

Zhu X, Kanai Y, Saito A, Kondo Y, Hirohashi S.

Pathol Int (Pathology International) 50 ( 12 ) 945 - 952 2000

Accepted, ISSN 13205463

The present study attempted to clarify the significance of aberrant expression of β-catenin protein and mutation of exon 3 of the β-catenin gene in renal and urothelial carcinogenesis. β-Catenin expression was examined immunohistochemically and mutation of the β-catenin gene was analyzed by polymerase chain reaction-single strand conformation polymorphism (SSCP) and direct sequencing. β-Catenin immunoreactivity was observed at the cell membrane in all 30 renal cell carcinomas (RCC) examined, and no RCC showed a mobility-shifted SSCP band. Of 46 transitional cell carcinomas (TCC) examined, there was reduced expression of β-catenin, as compared with its expression in non-cancerous transitional epithelium, in 22 cases (48%) and catenin accumulation in the nucleus in five cases (11%). Of four renal pelvis TCC examined, point mutation of exon 3 of the β-catenin gene at codon 45 resulting in amino acid substitution (Ser to Phe) was detected in one (25%). The incidence of reduced expression of β-catenin correlated significantly with the growth pattern (superficial type vs invasive type) of TCC (P < 0.05). These data indicate that: (1) aberrant β-catenin expression may be at least partly involved in urothelial carcinogenesis, but less significantly so in renal carcinogenesis, and (2) it may be associated with the progression of TCC showing invasive growth.

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Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis--A comprehensive study of loss of heterozygosity and microsatellite instability at 39 loci and DNA hypermethylation on 8 CpG islands in microdissected specimens from ---

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

Hepatology 32 970 - 979 2000

Accepted

Genetic instability and aberrant DNA methylation in chronic hepatitis and cirrhosis - A comprehensive study of loss of heterozygosity and microsatellite instability at 39 loci and DNA hypermethylation on 8 CpG islands in microdissected specimens from patients with hepatocellular carcinoma

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

Hepatology (Hepatology) 32 ( 5 ) 970 - 979 2000

ISSN 02709139

A study was conducted to examine the significance of genetic instability and aberrant DNA methylation during hepatocarcinogenesis. Genomic DNA was extracted from 196 microdissected specimens of noncancerous liver tissue that showed no marked histologic findings or findings compatible with chronic hepatitis or cirrhosis, and 80 corresponding microdissected specimens of hepatocellular carcinoma (HCC) from 40 patients. Loss of heterozygosity (LOH) and microsatellite instability (MSI) were examined by polymerase chain reaction (PCR) using 39 microsatellite markers, and DNA methylation status on 8 CpG islands was examined by bisulfite-PCR. In noncancerous liver tissues, LOH, MSI, and DNA hypermethylation were found in 15 (38%), 6 (15%), and 33 (83%) of 40 cases, respectively. The incidence of DNA hypermethylation in histologically normal liver was similar to that in chronic hepatitis and cirrhosis, although neither LOH nor MSI was found in histologically normal liver. In cancerous tissues, LOH, MSI, and DNA hypermethylation were found in 39 (98%), 8 (20%), and 40 (100%) of 40 cases, respectively. CpG islands of the p16 gene and methylated in tumor 1, 2, 12, and 31 clones were frequently methylated in cancerous tissues, although neither the thrombospondin-1 nor the human Mut L homologue (hMLH1) gene was methylated. Absence of silencing of the hMLH1 gene by DNA hypermethylation is consistent with the low incidence of MSI in HCCs. The results of this study indicate that LOH and aberrant DNA methylation contribute to hepatocarcinogenesis; DNA hypermethylation in particular, which precedes or may even cause LOH, is as an early event during hepatocarcinogenesis.

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Resection of liver metastases after pancreatoduodenectomy: Report of seven cases

Fujii K., Yamamoto J., Shimada K., Kosuge T., Yamasaki S., Kanai Y.

Hepato-Gastroenterology (Hepato-Gastroenterology) 46 ( 28 ) 2429 - 2433 1999.10

ISSN 01726390

There has been no English report of a long survivor after hepatectomy for metastasis from peri-ampullary malignancies, who had previously undergone pancreatoduodenectomy (PD) for primary disease. Herein, we report 7 patients of liver metastases who underwent 8 hepatectomies after PD for peri-ampullary malignancies. One patient whose liver metastasis was neuroendocrine tumor, survived 2 years and 6 months without recurrence after hepatectomy. Another patient who had 2 hepatectomies for metastasis from duodenal leiomyosarcoma survived for 3 years and 20 days after the first hepatectomy. Procedure of hepatectomy comprised 4 limited resections (including 1 second hepatectomy) and 4 lobectomies at the first resection. Hepatic inflow clamp was used in 6 out of 8 hepatectomies and 4 out of 8 hepatectomies did not require allogeneic blood transfusion. Bacterial contamination of the drained discharge from the cut surface of the liver, mostly representative of enteric organisms, was identified in all but 2 patients who were not examined. Subphrenic abscesses developed in 2 patients after removal of the drains. Thus, prophylactic use of abdominal drain is indispensable after hepatectomy for the patients with bilioenteric anastomosis.

DNA hypermethylation at the D17S5 locus and reduced HIC-1 mRNA expression are associated with hepatocarcinogenesis.

Kanai Y, Hui AM, Sun L, Ushijima S, Sakamoto M, Tsuda H, Hirohashi S.

Hepatology (Hepatology) 29 ( 3 ) 703 - 709 1999

Accepted, ISSN 02709139

To examine the significance of aberrant DNA methylation in hepatocarcinogenesis, the DNA methylation status at the D17S5 locus and mRNA expression of a candidate tumor suppressor gene, HIC-1 (hypermethylated-in- cancer), which was identified at the D17S5 locus, in primary hepatocellular carcinomas (HCCs) and their corresponding noncancerous liver tissues were assessed. DNA hypermethylation at the D17S5 locus was detected in 44% of the noncancerous liver tissues showing chronic hepatitis or cirrhosis, which are widely considered to be precancerous conditions, but was not observed in noncancerous liver tissues showing no remarkable histological findings. The incidence of DNA hypermethylation at this locus was significantly higher in HCCs (90%) than noncancerous liver tissues (P < .001). Loss of heterozygosity at the D17S5 locus, which was preceded by DNA hypermethylation at the same locus, was detected in 54% of HCCs. The HIC-1 mRNA expression level of noncancerous liver tissues showing chronic hepatitis or cirrhosis was significantly lower than that of noncancerous liver tissues showing no remarkable histological findings (P < .01), and that of HCCs was even lower than that of noncancerous liver tissues (P < .05). Poorly differentiated HCCs showed lower expression levels than well- to moderately differentiated HCCs. Mutation of the p53 gene may be involved in HIC-1 inactivation. Moreover, wild-type p53 did not overcome DNA hypermethylation at the D17S5 locus to activate HIC-1 in HCCs. These data suggest that aberrant DNA methylation at this locus and reduced HIC-1 mRNA expression participate in hepatocarcinogenesis during both early developmental stages and malignant progression of HCCs.

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Bilateral nonpalpable breast carcinomas in a patient with BRCA2 germ line mutation and past history of osteosarcoma.

Kuno T, Fukutomi T, Akashi-Tanaka S, Nanasawa T, Kanai Y, Tsuda H, Nomizu T, Miki Y.

Breast Cancer (Breast Cancer) 6 ( 1 ) 51 - 54 1999

Accepted, ISSN 13406868

A 34-year-old premenopausal woman developed asynchronous bilateral nonpalpable breast cancers at the age of 32 and 34 years. She had undergone amputation of her left lower leg because of osteosarcoma at the age of 16. Her mother had been diagnosed with breast cancer at the age of 45. The clinicopathological features of the two breast tumors in this patient closely resembled each other; both were nonpalpable, and detectable only by helical CT scan. Histologically, they consisted mainly of an intraductal component with small grade 3 invasive foci. In addition, both tumors estrogen receptor (ER) status was negative, and both were positive for c-erbB-2 protein on immunohistochemical staining. A missense germ line mutation of BRCA2 (exon 25 codon 3118; Met31 1 8Thr) was detected in this patient. These data may provide useful information on the carcinogenesis and biological behavior of breast cancers which develop in patients with BRCA2 germ line mutations.

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β-catenin accumulation and mutation of exon 3 of the β-catenin gene in hepatocellular carcinoma.

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

Jpn J Cancer Res (Japanese Journal of Cancer Research) 90 ( 12 ) 1301 - 1309 1999

Accepted, ISSN 09105050

A study was conducted to clarify the contribution of β-catenin accumulation and mutation of the β-catenin gene to hepatocarcinogenesis. β-Catenin accumulation was examined immunohistochemically in 38 paired samples of hepatocellular carcinoma (HCC) and corresponding non-cancerous liver tissue. Gene mutation was analyzed by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and direct sequencing using intronic primers encompassing exon 3. Neither accumulation nor mutation was detected in non-cancerous liver tissues that showed no remarkable histological features, chronic hepatitis or liver cirrhosis. Accumulation of β-catenin was seen in the nucleus, cytoplasm or cell membrane in 15 of 38 (39%) HCC samples, and gene mutation was seen in 9 of 38 (24%) HCC samples. Although there was a significant correlation between accumulation and mutation (P < 0.01), six HCCs without mutation also showed accumulation. Samples of early HCC showed neither accumulation nor mutation, and accumulation and mutation were each correlated significantly with portal vein tumor involvement (P < 0.05). The present results indicate that (1) mutation of exon 3 of the β-catenin gene can lead to β-catenin accumulation, although other mechanisms of accumulation may also operate in HCC, and (2) β-catenin accumulation and mutation of the β-catenin gene are not early events in hepatocarcinogenesis, and may be associated with the malignant progression of HCC.

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Synovial sarcoma, histologically mimicking primitive neuroectodermal tumor/Ewing's sarcoma at distant sites.

Masui F, Matsuno Y, Yokoyama R, Nakanishi Y, Hasegawa T, Kanai Y, Beppu Y, Hirohashi S, Fujii K, Shimoda T.

Jpn J Clin Oncol (Japanese Journal of Clinical Oncology) 29 ( 9 ) 438 - 441 1999

Accepted, ISSN 03682811

We report a case of synovial sarcoma (SS) showing unusual histology at distant sites. A 47-year-old man was aware of a tumor on the sole of his left foot. After preoperative chemotherapy with a diagnosis of SS, wide excision was performed. During postoperative chemotherapy, multiple tumorous lesions developed in the bone (including the whole spine) and both lungs. The patient died 1 year later. Histologically, the excised tumor of the foot showed a biphasic cellular pattern typical of SS, whereas at autopsy the bone and lung lesions were composed only of undifferentiated small round cells with cytoplasmic fibrillar processes. Homer-Wright rosettes were also observed. Immunohistochemically, 80% of the bone and lung tumor cells expressed MIC2 protein homogeneously. To clarify whether the bone and lung round cell tumors were metastatic lesions or second malignancies, especially primary primitive neuroectodermal tumor (PNET)/Ewing's sarcoma (ES), we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis of tumor type-specific fusion gene transcripts. The SYT/SSX fusion transcript was identified in both the foot and lung lesions, whereas the EWS/FLI1 transcript was not detected in either lesion. Therefore, we concluded that the multiple bone and lung tumors were poorly differentiated metastatic tumors, which arose from the SS of the foot. We also conclude that the identification of chimeric fusion transcripts can be successfully applied to poorly differentiated sarcomas and will help in the differential diagnosis of tumors that cannot be distinguished by conventional morphological examinations. Also, it should be remembered that cytoplasmic staining for MIC2 protein may occur in sarcomas other than PNET/ES. © 1999 Foundation for Promotion of Cancer Research.

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Microsatellite instability associated with hepatocarcinogenesis.

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

J Hepatol (Journal of Hepatology) 31 ( 3 ) 529 - 536 1999

Accepted, ISSN 01688278

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.

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Primary low-grade lymphoma of mucosa-associated lymphoid tissue of the urinary bladder: a case report with special reference to the use of ancillary diagnostic studies.

Ando K, Matsuno Y, Kanai Y, Sakamoto M, Fujimoto H, Narabayashi M, Tobisu K.

Jpn J Clin Oncol (Japanese Journal of Clinical Oncology) 29 ( 12 ) 636 - 639 1999

Accepted, ISSN 03682811

We report a case of primary low-grade B-cell lymphoma of the mucosa-associated lymphoid tissue (MALT) type of the urinary bladder. The patient, a 77-year-old woman, presented with a sense of urinary retention. An intravenous pyelogram and cystoscopy revealed a wide-based submucosal mass measuring 3 cm in the left wall of the urinary bladder. Histological findings of the tissue obtained by transurethral resection (TUR) showed a dense, monomorphic atypical lymphoid (centrocyte-like) infiltrate with reactive lymph follicles in the subepithelial tissue. Monocytoid and plasmacytoid features were readily evident in a population of these cells. Lymphoepithelial lesions involving the urothelium were also noticed in some areas. These features were strongly suggestive of primary low-grade lymphoma of the MALT type. The diagnosis was confirmed by immunohistochemical and flow cytometric studies, both of which showed a clear immunoglobulin restriction to lambda light chain and also by polymerase chain reaction-based assay using a formalin-fixed paraffin-embedded TUR tissue sample, which showed a clonal Ig heavy-chain gene rearrangement. Clinical staging procedures revealed that the tumor was localized in the urinary bladder. The patient has not received chemotherapy and is alive and well with no evidence of recurrence, 3 years after TUR. This case demonstrates that these ancillary tests are worth performing for confirmation of B-cell clonality in TUR tissue samples showing dense B-lymphocytic infiltration. © 1999 Foundation for Promotion of Cancer Research.

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Reduction of integrin β4 and enhanced migration on laminin in association with intraepithelial spreading of urinary bladder carcinomas.

Harabayashi T, Kanai Y, Yamada T, Sakamoto M, Ochiai A, Kakizoe T, Koyanagi T, Hirohashi S.

J Urol (Journal of Urology) 161 ( 4 ) 1364 - 1371 1999

Accepted, ISSN 00225347

Purpose: The aim of the present study was to investigate the biological and molecular basis of intraepithelial spreading (IES) of carcinomas in situ (CIS) of the urinary bladder, which were considered to be precursors of nodular invasive carcinomas. Materials and Methods: The propensity for IES of human transitional carcinoma cells was examined by inoculation into murine renal pelvis and urinary bladder, and the biological character of the cells with a high propensity for IES was explored in vitro. Results: Three of 6 cell lines exhibited a high propensity for IES. When cultured on laminin, these IES cells scattered, whereas 3 non-IES cells and 2 immortalized transitional epithelial cells did not. IES cells showed strong adhesiveness, haptotaxis and enhanced migration on laminin compared with both non-IES and immortalized transitional epithelial cells. In IES cells, expression of the integrin β4 subunit was markedly reduced and the integrin α6β1 complex was predominant compared with the integrin α6β4 complex. Transfection of IES cells with integrin β4 subunit cDNA inhibited their ability to migrate on laminin and their propensity for IES. In addition, expression of the integrin β4 subunit was reduced in surgically resected specimens of CIS of the urinary bladder. Conclusion: The results indicate that a reduction in integrin β4 plays a role in IES of CIS of the urinary bladder through enhanced migration on laminin.

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Disruption of E-cadherin-mediated cell adhesion systems in gastric cancers in young patients.

Saito A, Kanai Y, Maesawa C, Ochiai A, Torii A, Hirohashi S.

Jpn J Cancer Res (Japanese Journal of Cancer Research) 90 ( 9 ) 993 - 999 1999

Accepted, ISSN 09105050

The aim of this study was to elucidate the pathogenetic backgrounds of early-onset gastric cancers. Mutations of the E-cadherin and β-catenin genes were analyzed by subjecting microdissected cancer cells and corresponding non-cancerous epithelial cells obtained from 9 gastric cancer patients under 35 years old to polymerase chain reaction-single strand conformation polymorphism analysis. Somatic, but no germline, E-cadherin gene mutations were detected in 6 (67%) of the patients. The cancer cells of 2 patients were exon 9-deleted E-cadherin molecule-immunoreactive. Neither somatic nor germline mutations in exon 3 of the β-catenin gene were observed in any patient. One patient lacked β-catenin immunoreactivity and the cancer cells of 6 others showed cytoplasmic β-catenin immunoreactivity. The E-cadherin-mediated cell adhesion system in the cancer cells of all the patients examined appeared to be disrupted, indicating that somatically acquired dysfunction of this system plays an important role in early-onset diffuse-type gastric cancers. Helicobacter pylori infection was observed in 6 (67%) of our 9 patients, an incidence higher than the average in young Japanese individuals. Thus, early-onset gastric cancers may be attributabIe to environmental factors such as Helicobacter pylori infection.

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Reduced mRNA expression of the DNA demethylase, MBD2, in human colorectal and stomach cancers.

Kanai Y, Ushijima S, Nakanishi Y, Hirohashi S.

Biochem Biophys Res Commun (Biochemical and Biophysical Research Communications) 264 ( 3 ) 962 - 966 1999

Accepted, ISSN 0006291X

A study was performed to evaluate the significance of aberrations of the newly identified DNA demethylase, MBD2, in human carcinogenesis. Levels of expression of DNA demethylase mRNA were examined by reverse transcription followed by real-time quantitative detection of the PCR products in 32 samples of colorectal cancer tissue, 24 stomach cancers, and the corresponding noncancerous mucosae. DNA demethylase mRNA levels normalized with glyceraldehyde-phosphate dehydrogenase (GAPDH) mRNA were reduced in 31 (97%) of the 32 colorectal cancers and in 22 (92%) of the 24 stomach cancers when compared with the levels in the corresponding noncancerous mucosae. The average levels of DNA demethylase mRNA expression normalized with GAPDH mRNA in each of the colorectal (0.81 ± 0.55) and stomach (2.88 ± 0.23) cancers were significantly lower than in the noncancerous mucosae (1.90 ± 0.16 and 5.11 ± 0.34, respectively, p < 0.0001). There was no significant association between the DNA demethylase mRNA level and malignant potential in both colorectal and stomach cancers. These data suggest that reduced expression of DNA demethylase may play a role at a certain step of multistage carcinogenesis. Reduction of DNA demethylase mRNA expression may be, if anything, one of the early events of carcinogenesis, but may not participate in the malignant progression of tumors.

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Cloning and characterization of the human β<inf>4</inf>-integrin gene promoter and enhancers

Takaoka A., Yamada T., Gotoh M., Kanai Y., Imai K., Hirohashi S.

Journal of Biological Chemistry (Journal of Biological Chemistry) 273 ( 50 ) 33848 - 33855 1998.12

ISSN 00219258

The cell-surface adhesion molecule α6β4-integrin is a receptor for laminins and a component of hemidesmosomes. β4-Integrin expression is restricted to proliferating basal keratinocytes in the epidermis and is suppressed when differentiation commences. Altered β4-integrin expression levels correlate significantly with the aggressive behavior of cancers. In order to clarify the mechanisms that regulate transcription of the β4- integrin gene, we cloned its 5'-flanking region. This 5'-flanking region was found to have a high G + C content and not to contain either TATA or CAAT boxes. Nested delimitation and reporter analyses mapped a basal promoter to nucleotides -106 to +105, surrounding the most proximal transcription initiation site. Gel retardation and mutational analyses revealed that cooperation between AP1 and Ets, interacting with other factors, mediated the promoter activity. In addition to the promoter element, enhancer activity was found in the first intron (+1905/+3933) and in a sequence upstream of the promoter region (-414/-107). These findings should facilitate our understanding of the regulation of β4-integrin gene expression in processes such as cell growth and differentiation, apoptosis, and cancer development and metastasis.

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Reduced p27(Kip1) expression in hepatocellular carcinomas

Hui A., Sun L., Kanai Y., Sakamoto M., Hirohashi S.

Cancer Letters (Cancer Letters) 132 ( 1-2 ) 67 - 73 1998.10

ISSN 03043835

This study was designed to determine whether changes in p27(Kip1) expression are involved in human hepatocarcinogenesis. We examined the p27(Kip1) mRNA expression in hepatocellular carcinomas and surrounding non-cancerous liver tissue samples from 21 patients using a reverse-transcriptase polymerase chain reaction assay. The mean p27(Kip1) mRNA expression levels (ratio of p27(Kip1)/β-actin mRNA) were significantly lower in hepatocellular carcinomas than in non-cancerous liver tissues (0.49±0.24 versus 0.57±0.22, P<0.05). p27(Kip1) mRNA expression was reduced in 11 (52%) of the 21 hepatocellular carcinomas when compared with the surrounding non-cancerous liver tissues. These findings suggest that reduced p27(Kip1) expression may be at least partly responsible for human hepatocarcinogenesis. Copyright (C) 1998 Elsevier Science Ireland Ltd.

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Japanese late-onset breast cancer families: Their clinicopathological characteristics and absence of BRCA1 and BRCA2 germline mutations

Okumura T., Fukutomi T., Tsuda H., Kanai Y., Ushijima T., Akashi-Tanaka S., Nanasawa T.

Breast Cancer (Breast Cancer) 5 ( 3 ) 251 - 254 1998.07

ISSN 13406868

Background: The causes and pathologic and prognostic phenotypes of late-onset familial breast cancers are still unknown. The purpose of this study was to document the clinicopathological features of late-onset familial breast cancers using genetic testing of BRCA1 and BRCA2. Methods: We analyzed 11 breast cancers from 10 patients from 8 Japanese late-onset Breast cancer families. Results: The average age of the patients was 55 years (range 43 to 89). Bilateral occurrence was noted in 2 patients (8%). All the tumors were invasive ductal carcinomas, except for 1 case of invasive lobular carcinoma. Tumor size ranged from 0.8 cm to 7.8 cm (median 2.3 cm) and lymph node metastasis occurred in 6 of the 11 patients (55%). Six (55%) of the 11 tumors were histologically grade 2 and 5 (45%) were histologically grade 3. Estrogen receptor (ER) positivity was 80% (8/10). Overexpression of c-erbB-2 and p53 protein was detected in 18% (2/11) and 9% (1/11) of the tumors, respectively. Five patients from 4 families received genetic testing but all were negative for BRCA 1 and BRCA2 germline mutations. All the patients were alive after a median follow-up period of 32 months, except for 1 patient. Conclusions: In this study, no germline mutations of BRCA1 or BRCA2 were detected. However, there was a tendency towards ER-positive tumors, but the positivity of p53 protein was considered to be lower then that of sporadic tumors.

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Small polypoid lesions of the gallbladder. Differential diagnosis and surgical indications by helical computed tomography

Furukawa H., Kosuge T., Shimada K., Yamamoto J., Kanai Y., Mukai K., Iwata R., Ushio K.

Archives of Surgery (Archives of Surgery) 133 ( 7 ) 735 - 739 1998.07

ISSN 00040010

Objectives: To demonstrate the helical computed tomographic (CT) features of small polypoid lesions of the gallbladder and to establish a clinical strategy based on CT findings for the treatment of such lesions. Design: Validation cohort study. Setting: Tertiary care public hospital. Patients: Thirty-one patients with polypoid lesions of the gallbladder (≤3 cm) underwent CT followed by resection. Main Outcome Measure: The detectability of the lesions on both unenhanced and enhanced CT and the configuration of the lesions on enhanced CT were prospectively evaluated in comparison with the histopathological findings. Results: Unenhanced CT detected 14 (45%) of the 31 lesions, whereas enhanced CT detected all of the lesions. The detection rates of the neoplastic lesions (adenoma, adenocarcinoma, and metastatic tumor) and cholesterol polyps were 81% (13/16) and 7% (1/15), respectively (P<.001). Among the 20 lesions demonstrated as pedunculated, 6 (30%) were neoplastic, whereas 10 (91%) of the 11 lesions demonstrated as sessile were neoplastic (P<.001). When a lesion was demonstrated on unenhanced CT or its shape was sessile on enhanced CT, the case was diagnosed as a neoplastic lesion. The sensitivity, specificity, positive predictive value, negative predictive value, and overall accuracy of the CT diagnosis of the neoplastic lesions were 88% (14/16), 87% (13/15), 88% (14/16), 87% (13/15), and 87% (27/31), respectively. Conclusion: Computed tomography can differentiate neoplastic and nonneoplastic small polypoid lesions of the gallbladder and reliably identify the presence of neoplastic lesions that should be resected.

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Apocrine Carcinoma of the Breast: Clinicopathological Analysis and Histological Subclassification of 12 Cases.

Matsuo K, Fukutomi T, Tsuda H, Kanai Y, Tanaka SA, Nanasawa T.

Breast Cancer (Breast Cancer) 5 ( 3 ) 279 - 284 1998

Accepted, ISSN 13406868

Background: Apocrine carcinoma is rare and most of its clinicopathological features are still unknown. The purpose of this study was to identify the clinicopathological characteristics of apocrine carcinoma. Methods: We clinicopathologically analyzed apocrine breast carcinomas resected from 12 Japanese women and subclassified them histopathologically. Results: The average age of the patients at diagnosis was 60.1 years (range: 38-78). Tumor diameters ranged from 0.5 to 4.5 cm (median 2.4). Mammography revealed tumor shadows without microcalcifications in all patients. Two (17%) of the 12 patients were lymph node metastasis-positive, and lymphatic permeation by tumor cells was observed in 3 (25%). Apocrine carcinoma in this study could be classified into three subtypes according to predominant histopathological growth pattern: type I, intraductal spreading type (4 patients); type II, adenosis-associated type (3 patients); and type III, infiltrating type (5 patients). Types I and II showed no lymph node metastasis and had an excellent prognosis, whereas the infiltrating type was associated with lymph node metastasis and death from cancer. Estrogen receptors and progesterone receptors were positive in 17% (1/6) and 60% (3/5), respectively, of the tumors tested. Conclusions: In the present study, apocrine carcinoma of the breast was characterized by higher patient age and tumor shadows without microcalcifications on mammography. However, the tumors were heterogenous with regard to pattern of local spread.

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Local recurrence after conservative surgery without postoperative radiation for pure tubular carcinoma of the breast: a case report with reference to multifocality of tubular carcinoma.

Okano K, Fukutomi T, Tsuda H, Akashi-Tanaka S, Nanasawa T, Kanai Y.

Breast Cancer (Breast Cancer) 5 ( 2 ) 201 - 204 1998

Accepted, ISSN 13406868

A 49-year-old premenopausal woman with stage I breast carcinoma underwent left quadrantectomy with axillary dissection in 1992. The tumor was 0.7×0.5 cm. Histopathologically, this was a pure tubular carcinoma without lymph node metastasis or lymphatic or vascular invasion. Although the surgical margin was pathologically negative, atypical ductal hyperplasia was present close to the cut margin's edge. Neither adjuvant chemotherapy nor radiotherapy had been given after the operation. Approximately 5 years after the first surgery, she had a local recurrence in the vicinity of the operative wound. There was no clinical evidence of distant metastasis. A salvage mastectomy was performed. Histopathological examination revealed that the second tumor was an invasive ductal carcinoma, histological grade 2, with extensive intraductal component. It was difficult to determine whether this was a true in-breast recurrence or a second primary cancer. Overexpression of p53 and c-erbB-2 was observed in the second tumor. Estrogen receptor and progesterone receptor were both negative. No postoperative chemotherapy was given. Multifocality and atypical ductal hyperplasia were observed in 7 (87.5%) and 6 [75%) of 8 patients, respectively, with tubular carcinoma between 1991 and 1997 at the National Cancer Center Hospital. Coexisting disease associated with tubular carcinoma suggests that radiotherapy may be an important component of breast conservation treatment to prevent local recurrence in this type of tumor.

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Microinvasive breast carcinoma with extensive involvement of level III axillary lymph nodes: a case report.

Fujii K, Fukutomi T, Tsuda H, Akashi-Tanaka S, Nanasawa T, Kanai Y, Muramatsu Y.

Jpn J Clin Oncol (Japanese Journal of Clinical Oncology) 28 ( 1 ) 47 - 49 1998

Accepted, ISSN 03682811

A 44-year-old woman presented with a right huge axillary mass. Both mammography and ultrasonography revealed a primary cancer of 2.8 cm maximum diameter in the right breast and metastases in the axillary lymph nodes, both being confirmed by aspiration cytology as ductal carcinoma. Right standard radical mastectomy with level III axillary lymph node dissection was carried out. Pathologically, the tumor was diagnosed as ductal carcinoma in situ with microinvasion (DCISM), histologic grade 3. The area of stromal invasion measured 1 mm at its widest point. Sixteen of the 17 resected axillary lymph nodes contained metastases, including six level III lymph nodes. Immunohistochemical studies of the tumor revealed overexpression of p53 protein, but not that of c-erbB-2 protein. The frequency of lymph node metastases from DCISM is reported to be very low. Therefore, the present case with extensive involvement of level III lymph nodes was unusual.

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Breast cancer in Cowden's disease: a case report with review of the literature.

Tsubosa Y, Fukutomi T, Tsuda H, Kanai Y, Akashi-Tanaka S, Nanasawa T, Linuma G, Ushio K.

Jpn J Clin Oncol (Japanese Journal of Clinical Oncology) 28 ( 1 ) 42 - 46 1998

Accepted, ISSN 03682811

We report a case of invasive breast cancer in a 62-year-old female patient with Cowden's disease. A left modified radical mastectomy was performed and histopathology of the tumor showed invasive ductal carcinoma, histological grade 3, without lymph node metastasis. The patient had a past history of endometrial cancer at 55 but did not have a family history of malignant disease. Goiter was palpable but aspiration cytology revealed no malignancy. There were several papillomas on the oral mucosa and multiple papillomatous lesions on the right femur. Barium X-ray and endoscopic examination revealed multiple, small, hyperplastic polypoid lesions on the esophagus, stomach and rectum. Histopathology of the biopsy specimens from the esophagus and stomach showed acanthotic squamous epithelium and foveolar hyperplastic polyps. The patient was followed up closely to monitor the thyroid lesions and polyposis of the digestive tract. A total of 12 breast cancer patients who also had Cowden's disease have been reported in Japan and these cases are reviewed in this report.

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Expression of CD44 variants and its association with survival in pancreatic cancer.

Gotoda T, Matsumura Y, Kondo H, Saitoh D, Shimada Y, Kosuge T, Kanai Y, Kakizoe T.

Jpn J Cancer Res (Japanese Journal of Cancer Research) 89 ( 10 ) 1033 - 1040 1998

Accepted, ISSN 09105050

Since the CD44 variant 6(v6) molecule has been noted as a marker for tumor metastasis and prognosis in several tumors, we examined whether or not v6 is a useful marker for evaluating the prognosis of pancreatic cancer patients. In addition, we attempted to assess the clinicopathological implications for pancreatic cancer of the variant 2 (v2) isoform using a recently developed monoclonal antibody against a v2 epitope. The expression of CD44 variants was evaluated immunohistochemically in paraffin-embedded pancreatic cancer tissues from 42 patients who were confirmed surgically and histologically to have received curative resection. An indirect immunoperoxidase method was used with monoclonal antibodies against epitopes of the standard (CD44s) portion, v6 and v2. Protein expression data were evaluated statistically for any correlations with the length of survival or with histological parameters. The expression of CD44v6 and v2 was observed only in tumor cells, if at all. On the other hand, expression of total CD44 (including CD44v, as well as CD44s) was observed in both tumors and adjacent normal sites. Tumor tissue from 21 (50%) and 16 (38%) patients showed positive immunoreactivity with mAb 2F10 (anti-CD44v6) and mAb M23.6.1 (anti-CD44v2), respectively. The expression of CD44v6 and v2 was correlated with decreased overall survival (P = 0.0160 and P = 0.0125, respectively). A significant correlation was obtained between CD44v2 peptide expression and vessel invasion (P = 0.026). These results suggest that CD44v2 and CD44v6 may be useful markers for poor prognosis in curatively resected primary pancreatic cancer.

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Nonfunctioning islet cell tumors of the pancreas: clinical, imaging and pathological aspects in 16 patients.

Furukawa H, Mukai K, Kosuge T, Kanai Y, Shimada K, Yamamoto J, Mizuguchi Y, Ushio K.

Jpn J Clin Oncol (Japanese Journal of Clinical Oncology) 28 ( 4 ) 255 - 261 1998

Accepted, ISSN 03682811

Background: Nonfunctioning islet cell tumors (NFICTs) usually reach a large size prior to detection, at which stage patients have some symptoms or signs. Recently, NFICTs have been discovered in asymptomatic patients with increasing frequency owing to advances in diagnostic imaging techniques. This study investigated clinical, imaging and pathological features in recent cases of NFICT. Methods: The medical records, radiographs and pathological specimens of 16 patients with NFICT who were evaluated between April 1991 and March 1996 were reviewed. Results: Tumor sizes ranged from 0.8 to 17 cm (average, 5.2 cm). Five patients (31%) had some symptoms or signs at the time of diagnosis; however, the other 11 (69%) had no symptoms. Of 10 patients with a tumor of size 5 cm or less, nine were asymptomatic and all tumors were histologically benign. In contrast, in six patients with a tumor larger than 5 cm, four had some symptoms and five tumors were malignant. The detection rates of pancreatic tumor mass on ultrasonography and computed tomography were 94% (15/16) each. All 14 patients in whom the tumor was completely resected survived without recurrence; however, the remaining two patients with liver metastases died following recurrence. Conclusions: US and CT are useful in detecting NFICTs even if the tumor is small and the patient is asymptomatic. The detection of NFICTs of small size and their complete removal are essential for a successful cure.

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Small polypoid lesions of the gallbladder: differential diagnosis and surgical indications by helical computed tomography.

Furukawa H, Kosuge T, Shimada K, Yamamoto J, Kanai Y, Mukai K, Iwata R, Ushio K.

Arch Surg 133 735 - 739 1998

Accepted

Helical computed tomography in the diagnosis of portal vein invasion by pancreatic head carcinoma: usefulness for selecting surgical procedures and predicting the outcome.

Furukawa H, Kosuge T, Mukai K, Iwata R, Kanai Y, Shimada K, Yamamoto J, Ushio K.

Arch Surg (Archives of Surgery) 133 ( 1 ) 61 - 65 1998

Accepted, ISSN 00040010

Objective: To evaluate the use of helical computed tomography (CT) in diagnosing portal vein (PV) invasion by pancreatic head carcinoma and its usefulness in predicting outcome. Design: Validation cohort study. Setting: Tertiary care public hospital. Patients: Twenty-seven patients with carcinoma involving the pancreatic head were preoperatively studied with helical CT. All patients underwent resection. Main Outcome Measure: By quantifying the contact between the tumor and PV on helical CT, the relationship between them was classified into 1 of 4 types: type 1, visible fat layer between PV and the tumor; type 2, with the total PV circumference defined as 360°, contact between the tumor and PV was considered to be 90°or less; type 3, contact ranged between 91°and 180°; and type 4, contact greater than 180°. Helical CT results were compared with intraoperative observation, histological findings of the resected specimen, and postoperative course. Results: When helical CT showed type 3 or 4, the case was diagnosed as positive for PV invasion. Sensitivity, specificity, and overall accuracy were 83%, 100%, and 89% when compared with the intraoperative assessment, and 92%, 79%, and 85% with the histological assessment, respectively. One- and 2-year survival rates were 86% and 69% for type 1,100% and 75% for type 2, and 33% and 12% for type 3, respectively. The survival rates of patients with types 1 and 2 were significantly higher than that of those with type 3 (P<.05). All 3 patients with type 4 died within 9 months. Conclusions: Helical CT facilitates detection of PV invasion by pancreatic head carcinoma. The extent of PV involvement reflected the outcome after pancreatectomy.

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Epidermoid cyst in an intrapancreatic accessory spleen: CT and pathologic findings.

Furukawa H, Kosuge T, Kanai Y, Mukai K.

AJR Am J Roentgenol (American Journal of Roentgenology) 171 ( 1 ) 271 1998

Accepted, ISSN 0361803X

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Cloning and characterization of the human β4-integrin gene promoter and enhancers.

Takaoka AS, Yamada T, Gotoh M, Kanai Y, Imai K, Hirohashi S.

J Biol Chem 273 33848 - 33855 1998

Accepted

Reduced p27Kip1 expression in hepatocellular carcinomas.

Hui AM, Sun L, Kanai Y, Sakamoto M, aHirohashi S.

Cancer Lett 132: 67 - 73 1998

Accepted

DNA hypermethylation at the D17S5 locus is associated with gastric carcinogenesis.

Kanai Y, Ushijima S, Ochiai A, Eguchi K, Hui A, Hirohashi S.

Cancer Lett (Cancer Letters) 122 ( 1-2 ) 135 - 141 1998

Accepted, ISSN 03043835

The aim of this study was to elucidate the significance of aberrant DNA methylation in gastric carcinogenesis. The DNA methylation status at the D17S5 locus, at which a candidate tumor suppressor gene, HIC-1, was identified, of gastric cancers and non-cancerous gastric mucosae from 42 gastric cancer patients was examined by Southern blotting using a methylation-sensitive restriction enzyme. DNA hypermethylation was observed in 15, 13, 25 and 45% of the tissues showing no remarkable histological findings, chronic gastritis without intestinal metaplasia, intestinal metaplasia and gastric cancer, respectively. The incidence of DNA hypermethylation was significantly higher in gastric cancers than in non-cancerous gastric mucosae (P < 0.05). DNA hypermethylation was often accompanied by allelic loss at the same locus in gastric cancers. DNA hypermethylation at the D17S5 locus, which was even detected in precancerous conditions, including intestinal metaplasia, may play a role in gastric carcinogenesis.

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The E-cadherin gene is silenced by CpG methylation in human hepatocellular carcinomas.

Kanai Y, Ushijima S, Hui AM, Ochiai A, Tsuda H, Sakamoto M, Hirohashi S.

Int J Cancer (International Journal of Cancer) 71 ( 3 ) 355 - 359 1997

Accepted, ISSN 00207136

Our study was designed to clarify the significance of silencing the E- cadherin gene, which is located on 16q22.1, due to CpG methylation during hepatocarcinogenesis. The CpG methylation status of primary hepatocellular carcinomas (HCCs) and corresponding liver tissues showing chronic hepatitis or cirrhosis, which are widely considered to be precancerous conditions, were assessed by digesting DNA with methylation-sensitive and non-sensitive restriction enzymes. CpG methylation around the promoter region of the E- cadherin gene was detected in 46% of liver tissues showing chronic hepatitis or cirrhosis and 67% of HCCs examined. Immunohistochemical examination revealed reduced E-cadherin expression in 59% of HCCs examined. CpG methylation around the promoter region correlated significantly with reduced E-cadherin expression in HCCs (p < 0.05). CpG methylation around the promoter region, which increases during the progression from a precancerous condition to HCC, may participate in hepatocarcinogenesis through reduction of E- cadherin expression, resulting in loss of intercellular adhesiveness and destruction of tissue morphology.

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Histological prognostic parameters for adenocarcinoma of the pancreatic head. Proposal for scoring system for prediction of outcome.

Nakatsura T, Hasebe T, Tsubono Y, Ryu M, Kinoshita T, Kawano N, Konishi M, Kosuge T, Kanai Y, Mukai, K.

J Hep Bil Pancr Surg, 4 441 - 448 1997

Accepted

Insulin-dependent diabetic mastopathy: a case report.

Kuno T, Fukutomi T, Akashi-Tanaka S, Nanasawa T, Kanai Y, Tsuda H. I

Breast Cancer (Breast Cancer) 4 ( 3 ) 175 - 177 1997

Accepted, ISSN 13406868

We report a case of insulin-dependent diabetic fibrous mastopathy, which clinically simulated breast cancer. The patient is a 55-year-old woman with stony-hard breast lumps, who had been treated with insulin for diabetes mellitus. Mammmography revealed a highly-dense mass shadow in the left breast and ultrasonography detected hypoechoic lesions with acoustic shadows in both breasts, suggestive of malignancy. Excisional biopsy was performed for the left breast lump. On histopathological examination, this was fibrosis with dense lymphocytic infiltration around lobules, which is a characteristic feature of diabetic fibrous mastopathy.

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Intraductal papillary tumors and mucinous cystic tumors of the pancreas: clinicopathologic study of 38 cases.

Fukushima N, Mukai K, Kanai Y, Hasebe T, Shimada K, Ozaki H, Kinoshita T, Kosuge T.

Hum Pathol (Human Pathology) 28 ( 9 ) 1010 - 1017 1997

Accepted, ISSN 00468177

Clinicopathological features of 28 patients with intraductal papillary tumor (IDPT) and 10 patients with mucinous cystic tumor (MCT) of the pancreas were studied. Both IDPT and MCT showed papillary projections of the epithelium with abundant mucus secretion in the ectatic ducts or cystic spaces. The patients with IDPT comprised 19 men and 9 women with a mean age of 64.9 years. Three fourths of the IDPTs were located in the pancreatic head, and the mean tumor size was 9.5 cm. Local recurrence was observed in one patient, but none died of IDPT. In contrasts all patients with MCT were women, with a mean age of 49.4 years. None of the MCTs arose in the head, and the mean tumor size was 7.1 cm. One patient died of MCT, but all of the others survived without recurrence. Eight IDPTs and three MCTs showed invasion into the surrounding pancreatic tissue. Muconodular infiltration was mainly observed in five IDPTs and one MCTs and tubular infiltration in three IDPTs and two MCTs. A characteristic histological finding associated with the muconodular infiltration in IDPT was subepithelial 'mucin droplets' that appeared to represent a change in polarity of mucus secretion. The formation of such subepithelial 'mucin droplets' may he the initial step of muconodular infiltration in IDPT. Muconodular infiltration in IDPT appears different morphologically and biologically from the mucinous carcinoma subtype of conventional invasive ductal carcinorma.

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Metastatic renal cell carcinoma to the gallbladder: color Doppler sonography and CT findings.

Furukawa H, Mizuguchi Y, Kanai Y, Mukai K.

AJR Am J Roentgenol 169 1466 - 1467 1997

Accepted

Arteriovenous hemangioma of the gallbladder: CT and pathologic findings.

Furukawa H, Kanai Y, Mukai K, Yamasaki S.

AJR Am J Roentgenol 168 1383 1997

Accepted

Involvement of the anterior urethra in male patients with transitional cell-carcinoma of the bladder undergoing radical cystectomy with simultaneous urethrectomy.

Tobisu K, Kanai Y, Sakamoto M, Fujimoto H, Doi N, Horie S, Kakizoe T.

Jpn J Clin Oncol (Japanese Journal of Clinical Oncology) 27 ( 6 ) 406 - 409 1997

Accepted, ISSN 03682811

Histological tumor extension into the prostate, urethra and urethra was examined in 52 specimens obtained by cystourethrectomy for transitional cell carcinoma of the bladder with one or more risk factors for urethral involvement. In four (21.1%) of 19 patients with diffuse carcinoma in situ in the bladder extending to the internal urethral orifice and prostatic urethra, the anterior urethra was affected by transitional cell carcinoma, including one specimen with invasion into the corpus spongiosum. However, none of 33 patients without these findings had synchronous anterior urethral transitional cell carcinoma involvement. It is recommended that patients with diffuse carcinoma in situ extending into the prostatic urethra undergo careful preoperative assessment of the anterior urethra before cystectomy. In other cases, the anterior urethra can be preserved and used for orthotopic neobladder replacement.

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Differentiating agent, hexamethylene bisacetamide, inhibits neointimal formation after vascular injury.

Ishikawa S, Hori S, Kusuhara M, Kanai Y, Handa S, Yamaguchi K, Hosoda Y, Ogawa, S.

Coron Artery Dis (Coronary Artery Disease) 8 ( 1 ) 28 - 32 1997

Accepted, ISSN 09546928

Background: Hexamethylene bisacetamide (HMBA), a potent differentiating agent of transformed cell lines, has been reported to affect phenotypic modulation and suppress proliferation of vascular smooth muscle cells (VSMCs). The effects of HMBA on the growth of VSMCs were studied in the rat carotid injury model. Methods: HMBA was added to culture medium of VSMCs explanted from aorta of Sprague Dawley rats and the cell number was counted after 5 days of incubation. The carotid artery of Sprague Dawley rats was denuded with a balloon embolectomy catheter. The rats were treated with continuous infusion of HMBA (1 g/day) or saline for 7 days after injury. Results: HMBA inhibited VSMC proliferation in a dose-dependent manner (at a HMBA concentration of 1-5 mM). Plasma concentration of HMBA during continuous infusion was in the range 0.8-1.0 mM. Immunohistochemistry of proliferating cell nuclear antigan showed that HMBA inhibited VSMC proliferation after vascular injury. Cell number in the intima and neointimal thickening were significantly reduced in HMBA-treated rats at 14 days after injury. Conclusions: Systemic administration of HMBA inhibited VSMC proliferation in the rat carotid injury model.

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Temporal alterations of endothelial-vasodilator functions in lung injury induced by monocrotaline.

Yamaguchi K, Kanai Y, Asano K, Takasugi T, Tanaka T, Yasuoka M, Hosoda Y.

Respir Physiol (Respiration Physiology) 107 ( 1 ) 47 - 58 1997

Accepted, ISSN 00345687

To assess the time course of alterations in pulmonary endothelial vasodilator functions during pathological development of pulmonary hypertension (PH) and right ventricular hypertrophy (RVH), we examined changes in serotonin (5-HT) removal rates and the production of prostacyclin (PGI2) and nitric oxide (NO) in isolated rat lungs harvested at various times after single exposure to monocrotaline (MCT). We assessed the generation of vasodilator substances under conditions of both the absence and the presence of 5-HT in lungs perfused with blood-free solution. Major findings included: (i) remodeling of the pulmonary vasculature associated with RVH evident 14 days after MCT injection; (ii) the capacity for 5-HT removal was suppressed at day 1 and 7 but had been restored by day 14 after MCT exposure; (iii) basal PGI2 production in the absence of 5-HT was augmented at day 1 but had returned to control levels in lungs harvested 7 or 14 days postinjection of MCT; (iv) PGI2 production evoked by 5-HT was suppressed in MCT lungs obtained at all time points examined; (v) basal NO production was suppressed at day 1 but enhanced at day 7 and 14 in MCT lungs; (vi) NO production elicited by 5-HT stimulation in 1-day-MCT lungs was obviously suppressed while that in 7- and 14-day-MCT lungs had been restored to the control level. These findings suggest that transitional changes in endothelial functions including 5-HT removal and production of vasodilators in MCT lungs do not follow the same time course.

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

Hui AM, Kanai Y, Sakamoto M, Tsuda H, Hirohashi S.

Hepatology (Hepatology) 25 ( 3 ) 575 - 579 1997

Accepted, ISSN 02709139

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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Increased DNA methyltransferase expression is associated with an early stage of human hepatocarcinogenesis.

Sun L, Hui AM, Kanai Y, Sakamoto M, Hirohashi S.

Jpn J Cancer Res (Japanese Journal of Cancer Research) 88 ( 12 ) 1165 - 1170 1997

Accepted, ISSN 09105050

The present study was designed to determine whether changes in DNA methyltransferase (DNA MTase) expression are involved in hepatocarcinogenesis. We examined DNA MTase expression in normal liver tissue (with no remarkable histological findings), liver tissue showing chronic hepatitis or cirrhosis, which are generally thought to be precancerous conditions, and hepatocellular carcinomas (HCCs) using the reverse-transcriptase polymerase chain reaction assay. DNA MTase mRNA levels were significantly higher in liver tissue showing chronic hepatitis and cirrhosis (DNA MTase mRNA/β-actin mRNA ratio = 0.30 ± 0.22, n = 24, P < 0.01) than in normal liver tissue either from patients with liver metastatic lesions of colonic cancer (0.14 ± 0.05, n = 6) or from patients with HCCs (0.16 ± 0.07, n = 3). DNA MTase mRNA levels were even higher in HCC tissue (0.34 ± 0.18, n = 29). These results suggest that increased DNA MTase expression may be an early event during hepatocarcinogenesis. DNA MTase is a potential target for HCC preventive therapy.

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DNA hypermethylation at the D17S5 locus in non-small cell lung cancers: its association with smoking history.

Eguchi K, Kanai Y, Kobayashi K, Hirohashi S.

Cancer Res (Cancer Research) 57 ( 21 ) :4913 - 4915 1997

Accepted, ISSN 00085472

The aim of this study was to examine the association between DNA hypermethylation and clinicopathological features of non-small cell lung cancers (NSCLCs). The DNA methylation status at the D17S5 loci, at which a candidate tumor suppressor gene, HIC-1 (hypermethylated in cancer), was identified, of 51 paired tumor and nontumorous lung tissue specimens from NSCLC patients were examined by Southern blot analysis, using a methylation- sensitive restriction enzyme. DNA hypermethylation at this locus was found in 17 (33%) tumors and 16 (31%) nontumorous lung tissues. DNA in hypermethylation at this locus occurred more frequently in poorly than in well-differentiated tumors, especially in adenocarcinomas, and correlated significantly with the differentiation grade (P = 0.01). DNA hypermethylation at the D17S5 locus correlated significantly with the loss of heterozygosity at this locus in tumors (P = 0.01). The incidence of DNA hypermethylation was significantly higher in smokers than those who had never smoked in both tumors and nontumorous lung tissues (P = 0.03 and P = 0.01, respectively). These results suggest that DNA hypermethylation at the D17S5 locus may play a role in the development of NSCLCs in cigarette smokers.

Histological prognostic parameters for adenocarcinoma of the pancreatic head. Proposal for a scoring system for prediction of outcome

Kanai Y.

Journal of Hepato-Biliary-Pancreatic Surgery (Journal of Hepato-Biliary-Pancreatic Surgery) 4 ( 4 ) 441 - 448 1997

ISSN 09441166

To find new histopathological prognostic parameters for adenocarcinoma of the pancreatic head, 27 of these carcinomas, at stage IV according to the Japan Pancreas Society, which had been curatively resected, were histologically examined. In addition to previously recognized histological prognostic parameters, sections were examined for the following factors: fibrotic focus (FF), direct invasion of the tumor into the lymph node (DILN), and tumor necrosis (TN). Frequency of tumor recurrence or death was higher in patients with any one of the three new factors than in those without any of these factors. To develop an accurate system for predicting the outcome, the presence or absence of each factor and lymphatic permeation was given a score of 1 (present) or 0 (absent), and the total score was than calculated for each patient. All patients with a score of 0 were alive without tumor recurrence, at a mean follow-up of 20 months, whereas all seven patients with a score of 4 had experienced tumor recurrence, and six died subsequently. The results of this study demonstrated that FF, DILN, and TN are good histological parameters of ductal adenocarcinoma of the pancreatic head, and that the scoring system proposed in this paper is useful for prediction of the outcome of the disease. © Springer-Verlag 1997.

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Inactivation of p16(INK4) in hepatocellular carcinoma

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

Hepatology (Hepatology) 24 ( 3 ) 575 - 579 1996.09

ISSN 02709139

We analyzed the p16(INK4) status of 6 hepatocellular carcinoma (HCC) cell lines and 32 primary HCC tumors, including 9 early-stage tumors, to determine whether p16(INK4) tumor-suppressor gene inactivation participates in hepatocarcinogenesis. p16(INK4) 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 p16(INK4) 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 p16(INK4) homozygous deletion and mutation nor loss of p16(INK4) 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 p16(INK4) mRNA was not detected. These results suggest that p16(INK4) in HCC is inactivated predominantly by posttranscriptional regulation rather than by genomic aberrations and lack of transcription.

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Aberrant DNA methylation on chromosome 16 is an early event in hepatocarcinogenesis.

Kanai Y, Ushijima S, Tsuda H, Sakamoto M, Sugimura T, Hirohashi S.

Jpn J Cancer Res 87: 1210-1217, 1996. (Japanese Journal of Cancer Research) 87 ( 12 ) 1210 - 1217 1996

Accepted, ISSN 09105050

In order to clarify the significance of DNA methylation in both earlier and later stages of hepatocarcinogenesis, the DNA methylation state on chromosome 16, on which loss of heterozygosity (LOH) has frequently been detected in human hepatocellular carcinomas (HCCs), was examined. DNA from primary HCCs and tissues showing chronic hepatitis and liver cirrhosis, which are considered to be precancerous conditions, was analyzed by digestion with methylation-sensitive and non-sensitive restriction enzymes. DNA hypermethylation at the D16S32, tyrosine aminotransferase (TAT) and D16S7 loci and hypomethylation at the D16S4 locus were detected in 18%, 58%, 20% and 48% of examined HCCs, respectively. Aberrant DNA methylation occurred more frequently in advanced HCCs than in early HCCs. Moreover, DNA hypermethylation at the D16S32, TAT and D16S7 loci was frequently observed in chronic hepatitis and liver cirrhosis. The incidence of DNA hypermethylation was higher than that of LOH (42% at the TAT locus). These data suggest that DNA hypermethylation might predispose the locus to allelic loss. Aberrant DNA methylation is a significant change which may participate in the early developmental stages of HCCs.

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Formation of focal adhesion and spreading of polarized human colon cancer cells in association with tyrosine phosphorylation of paxillin in response to phorbol ester.

Sakamoto M, Ino Y, Ochiai A, Kanai Y, Akimoto S, Hirohashi S.

Lab Invest (Laboratory Investigation) 74 ( 1 ) 199 - 208 1996

ISSN 00236837

Regulation of cell adhesion systems is involved in both normal development and the invasive behavior of carcinomas. We examined alterations of cell morphology and adhesion molecules in response to phorbol ester treatment of the SW1116 colon cancer cell line, which forms well-organized dome-like tubular structures in culture. 12-O-Tetradecanoylphorbol-13 acetate (TPA) induced rapid spreading of cancer-cell colonies through formation of focal adhesion and disappearance of adherens junctions. Immunologic analyses demonstrated that tyrosine-phosphorylated proteins were concentrated at focal adhesions, and that tyrosine phosphorylation of two proteins, paxillin and an unidentified 130-kd protein, was significantly increased. Tyrosine phosphorylation of paxillin was detectable within 15 min after TPA treatment, when only lamellipodia had extended from the colony, and in cells treated with blocking antibodies against integrins β1 and β5, which strongly inhibited spreading and disorganization while preserving adherens junctions. The level of paxillin phosphorylation correlated well with the degree of morphologic change induced by low-dose TPA, and the dephosphorylation occurred before reversion of morphology upon removal of TPA. These findings suggest that the TPA signal was transduced to the tyrosine phosphorylation of paxillin strongly associated with formation of focal adhesion, and that this in turn induced dysfunction of the cadherin system and caused spreading and disorganization of the tubular structure. The mechanism responsible for disruption of the cadherin system at adherens junctions was not clear, but the transition of β-catenin into nuclei corresponded to the disappearance of its signal along areas of cell-cell contact. This SW1116 model provides a good system for studying the molecules involved in transient regulation and crosstalk between the cell-cell and cell-substratum adhesion systems, which may explain the mechanism of invasion of well-differentiated human carcinomas.

Serous cystadenoma of the pancreas communicating with a pancreatic duct.

Furukawa H, Takayasu K, Mukai K, Kanai Y, Kosuge T, Mizuguchi Y, Ushio K.

Int J Pancreatol (International Journal of Pancreatology) 19 ( 2 ) 141 - 144 1996

Accepted, ISSN 01694197

Conclusion: To differentiate serous cystadenoma from other cystic lesions communicating with the pancreatic duct, careful radiological examination is necessary. Background: Communication between the cystic cavity and the pancreatic duct in an ordinary serous cystadenoma is uncommon, although it is not uncommon in other cystic lesions, such as pseudocyst, mucinous cystadenoma/adenocarcinoma, and intraductal papillary tumor. Methods: A serous cystadenoma of the pancreas communicating with main pancreatic duct in a 76-yr-old male is reported. Results: The communication was preoperatively demonstrated by endoscopic retrograde cholangiopancreatography and confirmed by histopathological examination of the resected specimen.

Late contrast-enhanced CT for small pancreatic carcinoma: delayed enhanced area on CT with histopathological correlation.

Furukawa H, Takayasu K, Mukai K, Kanai Y, Inoue K, Kosuge T, Ushio, K.

Hepatogastroenterology (Hepato-Gastroenterology) 43 ( 11 ) 1230 - 1237 1996

Accepted, ISSN 01726390

Purpose: To determine the appearance of pancreatic adenocarcinoma on delayed contrast enhanced computed tomography (CT) and verify the diagnostic significance. Patients and Methods: Twenty-two surgically resected lesions of pancreatic adenocarcinoma were studied with dynamic CT and findings were compared with those on histopathology. Results: Ten 45% of 22 pancreatic adenocarcinoma demonstrated masses on unenhanced CT, while 21 (95%) demonstrated on early contrast-enhanced CT (early CT), and 15 (68%) demonstrated masses on delayed contrast-enhanced CT (late CT). Pancreatic mass was demonstrated as a low-density area on unenhanced and early CT, but mass density varied on late CT. Delayed enhancement of the mass was predominantly observed in small lesions; 3 (75%) of 4 lesions 2 cm or smaller were detected as high-density areas. Histologically, delayed enhanced lesions showed severe or moderate degrees of fibrosis. Conclusion: Late CT is useful for the detection of pancreatic adenocarcinoma with fibrosis as a high-density area, especially in small lesions.

E-cadherin gene mutations in signet ring cell carcinoma of the stomach.

Muta H, Noguchi M, Kanai Y, Ochiai A, Nawata H, Hirohashi S.

Jpn J Cancer Res (Japanese Journal of Cancer Research) 87 ( 8 ) 843 - 848 1996

Accepted, ISSN 09105050

To clarify the significance of E-cadherin gene alterations in the development of diffuse-type adenocarcinoma of the stomach, we analyzed mutations of the E-cadherin gene using the polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) method followed by direct sequencing. Twenty-two signet ring cell carcinomas of the stomach (10 intramucosal and 12 advanced cancers) were examined. Genomic DNA was extracted separately from cancerous and non-cancerous tissues and PCR-SSCP analysis was performed on exons 5 to 9 and the adjacent 30- to 40 base pair intron sequences of the E-cadherin gene. Mobility shifts were found in 2 of the 10 intramucosal cancers. In 2 of the 12 advanced cancers, abnormalities of the E-cadherin gene were observed in intramucosal lesions as well as in deeply invaded areas. These results indicated that E-cadherin gene mutations are an early event in the development of signet ring cell carcinoma of the stomach. Direct sequencing revealed that the locations of mutations of the E-cadherin gene included the branch point sequence in the intron which is responsible for RNA splicing. Reverse transcriptase-PCR demonstrated aberrant RNA splicing in a case with a branch point mutation, suggesting that branch point mutations play an important role in functional modifications of E-cadherin in signet ring cell carcinoma of the stomach.

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Inactivation of p16INK4 in hepatocellular carcinoma.

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

Hepatology 24 575 - 579 1996

Accepted

Dominant negative inhibition of the association between β-catenin and c-erbB-2 by N-terminally deleted β-catenin suppresses the invasion and metastasis of cancer cells.

Shibata T, Ochiai A, Kanai Y, Akimoto S, Gotoh M, Yasui N, Machinami R, Hirohashi, S.

Oncogene (Oncogene) 13 ( 5 ) 883 - 889 1996

ISSN 09509232

Aberrant tyrosine phosphorylation of β-catenin inactivates the E-cadherin-mediated cell adhesion and invasion suppressor system in cancer cells. Elucidation of the association between β-catenin and c-erbB-2 protein prompted us to investigate whether interference with this interaction can change the invasive phenotype. In a human gastric cancer cell line, TMK-1, N-terminally deleted β-catenin, which binds to c-erbB-2 but not to cadherin, inhibited the association between endogenous β-catenin and c-erbB-2 protein, and suppressed the tyrosine phosphorylation of β-catenin. Cells expressing truncated β-catenin exhibited markedly reduced invasiveness in vitro and peritoneal metastasis in vivo, and developed an epithelial morphology. These results suggest that tyrosine phosphorylation of β-catenin regulated by c-erbB-2 protein may play an important role in the invasion, metastasis and morphogenesis of cancer cells and that inhibition of the aberrant tyrosine phosphorylation of β-catenin effectively prevents invasion and metastasis of cancer cells.

c-erbB-2 gene product directly associates with β-catenin and plakoglobin.

Kanai Y, Ochiai A, Shibata T, Oyama T, Ushijima S, Akimoto S, Hirohashi S.

Biochem Biophys Res Commun (Biochemical and Biophysical Research Communications) 208 ( 3 ) 1067 - 1072 1995

Accepted, ISSN 0006291X

Association of the c-erbB-2 oncogene product with the cadherin-catenin complex has been demonstrated in human cancer cell lines. Although β-catenin and plakoglobin have been proven to be crucial for the association, no previous study has shown whether the interactions are direct or indirect. In the present study, the c-erbB-2 gene product was shown by far-Western blotting analysis to associate directly with both β-catenin and plakoglobin through its cytoplasmic domain core region, which showed extensive homology with epidermal growth factor receptor. These data suggest that c-erbB-2-induced signaling is also directly liked to the cadherin-mediated cell adhesion and "invasion-suppressor" system through β-catenin and plakoglobin in cancers. © 1995 Academic Press. All rights reserved.

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Expression of N-cadherin and α-catenin in astrocytomas and glioblastomas.

Shinoura N, Paradies NE, Warnick RE, Chen H, Larson JJ, Tew JJ, Simon M, Lynch RA, Kanai Y, Hirohashi S, and et al.

Br J Cancer (British Journal of Cancer) 72 ( 3 ) 627 - 633 1995

Accepted, ISSN 00070920

We examined levels of mRNA and protein for N-cadherin, the predominant cadherin in neural tissues, and mRNA levels for the cadherin-associated protein, alpha-catenin, in a series of gliomas and in glioblastoma cell lines. mRNA levels for N-cadherin and alpha-catenin were significantly higher in glioblastomas than in low-grade astrocytomas or normal brain, while the levels of intact N-cadherin protein were similar in glioblastomas, low-grade astrocytomas and brain. In addition, there was no consistent relationship between invasiveness and expression of N-cadherin and alpha-catenin in highly invasive vs minimally invasive tumours within the same histopathological grade. To assess further the relationship between cadherin expression and neural tumour invasion, we measured N-cadherin expression, calcium-dependent cell adhesion and motility of several glioblastoma cell lines. While all N-cadherin-expressing lines were adhesive, no correlation was seen between the level of N-cadherin expression and cell motility. Together, these findings imply that, in contrast to the role played by E-cadherin in carcinomas, N-cadherin does not restrict the invasion of glioblastomas. © 1995 Stockton Press. All rights reserved.

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Silencing of the E-cadherin invasion-suppressor gene by CpG methylation in human carcinomas.

Yoshiura K, Kanai Y, Ochiai A, Shimoyama Y, Sugimura T, Hirohashi S.

Proc Natl Acad Sci USA (Proceedings of the National Academy of Sciences of the United States of America) 92 ( 16 ) 7416 - 7419 1995

Accepted, ISSN 00278424

E-Cadherin, a cell adhesion molecule, which plays a key role in maintaining the epithelial phenotype, is regarded as an invasion-suppressor gene in light of accumulating evidence from in vitro experiments and clinical observations. In an attempt to clarify the mechanism responsible for inactivation of this gene in carcinomas, we investigated the methylation state around the promoter region by digestion of DNA with the methylation- sensitive restriction enzyme Hpa II, as CpG methylation of the promoter has been postulated to be a mechanism of transcriptional inactivation of some genes. We found that E-cadherin expression-negative carcinoma cell lines were accompanied by the hypermethylation state, whereas E-cadherin-positive cell lines were not. Furthermore, treatment of E-cadherin-negative carcinoma cells with the demethylating agent 5-azacytidine resulted in reexpression of the gene and reversion of scattered spindle-shaped cells to cells with epithelial morphology. These results suggest that hypermethylation around the promoter may be a mechanism of E-cadherin inactivation in human carcinomas and that treatment of E-cadherin-inactivated cells with a demethylating agent may cause gene expression reversion leading to epithelial morphogenesis with acquisition of the homophilic cell-cell adhesive property.

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Analysis of the neurofibromatosis 2 gene in human breast and hepatocellular carcinomas.

Kanai Y, Tsuda H, Oda T, Sakamoto M, Hirohashi S.

Jpn J Clin Oncol (Japanese journal of clinical oncology) 25 ( 1 ) 1 - 4 1995

Accepted, ISSN 03682811

The neurofibromatosis 2 gene (NF2) mapped to chromosome 22q is a recently isolated tumor suppressor gene that participates in the tumorigenesis of cells of embryonic neural crest origin. The structural similarities between the NF2 product, merlin-schwannomin, and the band 4.1 family raise the possibility that merlin may participate in a wide range of cell activities, in which the cytoskeleton latticework has important roles to play. In order to examine the significance of NF2 in general carcinogenesis, comprehensive analyses of 68 cases of breast carcinoma, which shows frequent loss of heterozygosity at chromosome 22, and 48 hepatocellular carcinomas of different histological grade were performed. No mutation was detected by the polymerase chain reaction-single-strand conformation polymorphism method for six exons of NF2 in any of the cases examined, suggesting that NF2 may be less important in the tumorigenesis of breast and liver cancers than in that of cancers originating from the neural crest.

Point Mutation of the E–Cadherin Gene in Invasive Lobular Carcinoma of the Breast

Kanai Y., Oda T., Tsuda H., Ochiai A., Hirohashi S.

Japanese Journal of Cancer Research (Japanese Journal of Cancer Research) 85 ( 10 ) 1035 - 1039 1994.10

ISSN 09105050

Reduced or heterogeneous expression of E–cadherin has been demonstrated immunohistochemically in poorly differentiated carcinoma, which frequently shows weak intercellular adhesiveness and marked invasiveness. In vitro, not only reduced expression but also structural abnormalities of E–cadherin have been observed in human carcinoma cell lines which grow in a loosely adhering manner. To clarify the participation of structural abnormalities of E–cadherin in cancer invasion in vivo, sequence abnormalities were examined in the cadherin domain (exons 5, 6, 7 and 8) including the region essential for E–cadherin specific binding, using the polymerase chain reaction–single–strand conformation polymorphism method and direct sequencing in invasive lobular carcinoma of the breast, in which cancer cells become detached from each other and invade the stroma in a particularly scattered pattern. In 2 (10%) of the 20 cases examined, an identical sequence abnormality was detected in E–cadherin exon 7, i.e. a point mutation of codon 315 (AAT to AGT) which resulted in a single ainino acid substitution (asparagine to serine). This mutation may abolish the E–cadherin–mediated cell–cell adhesion and be at least partly responsible for the weak intercellular adhesiveness and scattered histological pattern of the tumor. Copyright © 1994, Wiley Blackwell. All rights reserved

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Role of serotonin in the progression of pulmonary hypertension

Hosoda Y., Kanai Y.

[Rinshō ketsueki] The Japanese journal of clinical hematology ([Rinshō ketsueki] The Japanese journal of clinical hematology) 35 ( 4 ) 376 - 380 1994.04

ISSN 04851439

From 0.5 to 3 days after subcutaneous injection of monocrotaline (MCT) 60 mg/kg, prominent accumulation of platelets in the pulmonary capillaries accompanied with significant elevation of the plasma serotonin level was observed. To clarify the role of serotonin, the in vivo and in vitro effects of the selective 5-HT2 receptor antagonist, DV-7028, on MCT-induced pulmonary hypertension were studied. Oral administration of DV-7028 (10 mg/kg, twice daily) significantly suppressed the MCT-induced elevation of pulmonary arterial pressure, right ventricular hypertrophy and medial thickening of the muscular-type pulmonary arteries which occurred 23 days after MCT administration. The plasma level achieved by oral administration of 10 mg/kg DV-7028 was more than 10(-7) M. The hyperreactivity to serotonin in isolated pulmonary artery segments from MCT-treated rats was significantly reduced by DV-7028 (10(-7) M). The present study suggests that serotonin, released from platelets and accumulated in pulmonary capillaries, contributes to the initiation and/or progression of pulmonary hypertension in MCT-treated rats.

Point mutation of the E-cadherin gene in invasive lobular carcinoma of the breast.

Kanai Y, Oda T, Tsuda H, Ochiai A, Hirohashi, S.

Jpn J Cancer Res 85 1035 - 1039 1994

Accepted

Alterations of the cadherin-catenin cell adhesion system in cancers.

Kanai Y, Oda T, Shimoyama Y, Ochiai A, Oyama T, Yoshiura K, Akimoto S, Yamada T, Hirohashi, S.

Princess Takamatsu Symp (Princess Takamatsu symposia) 24 51 - 62 1994

The E-cadherin-mediated cell adhesion system acts as an "invasion suppressor" system, which is widely considered to be inactivated when the expression of E-cadherin is reduced and/or heterogeneous. To further investigate the molecular mechanisms responsible for dysfunction of this system in cancers, we examined human carcinoma cell lines lacking tight cell-cell adhesion. In KATO-III, established from stomach cancer, a point mutation of the E-cadherin gene resulted in a mRNA splicing error and markedly reduced E-cadherin expression. In another stomach cancer cell line, MKN 45, an 18-bp deletion of the E-cadherin gene caused a mRNA splicing error and a 4-amino-acid deletion, which was considered to alter the conformation around the key Ca(2+)-binding motif. In these two cell lines, the wild-type allele of the E-cadherin locus, which was assigned to chromosome 16q, was lost. Also in vivo, we found mutation of E-cadherin in breast cancers, where allele loss on chromosome 16 has frequently been reported. Thus, dysfunction of E-cadherin could be caused by a combination of the loss of one allele and a mutation in the remaining allele. Homologous deletion of part of the alpha-catenin gene, resulting in markedly reduced expression, was observed in a human lung cancer cell line, PC9. Recently, we also found mutations of beta-catenin in human carcinoma cell lines. These findings indicate the possible involvement of genetic abnormalities of various components in inactivation of the E-cadherin-mediated "invasion suppressor system" in cancers.

c-erbB-2 gene product associates with catenins in human cancer cells.

Ochiai A, Akimoto S, Kanai Y, Shibata T, Oyama T, Hirohashi S.

Biochem Biophys Res Commun (Biochemical and Biophysical Research Communications) 205 ( 1 ) 73 - 78 1994

Accepted, ISSN 0006291X

Phosphorylation of β-catenin, an intracytoplasmic cadherin-binding protein, causes disruption of the cadherin-mediated cell adhesion system in cancer cells. A 185-kDa phosphorylated protein, identified as the c-erbB-2 gene product, was co-immunoprecipitated with the E-cadherin-catenin complex. Association of the c-erbB-2 gene product with the cadherin-catenin complex was proved to be mediated through β-catenin and plakoglobin using an in vitro protein-protein precipitation system. These results indicate that the c-erbB-2 gene product associates with catenins and may regulate the cell adhesion and invasive growth of cancer. © 1994 Academic Press, Inc.

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A truncated β-catenin disrupts the interaction between E-cadherin and α-catenin: a cause of loss of intercellular adhesiveness in human cancer cell lines.

Oyama T, Kanai Y, Ochiai A, Akimoto S, Oda T, Yanagihara K, Nagafuchi A, Tsukita S, Shibamoto S, Ito F, and et al.

Cancer Res (Cancer Research) 54: ( 23 ) 6282 - 6287 1994

Accepted, ISSN 00085472

Cadherin cell adhesion molecules play an essential role in creating tight intercellular association and are considered to work as an invasion suppressor system of cancer cells. They form a molecular complex with catenins, a group of cytoplasmic proteins including α- and β-catenins. While α-catenin has been demonstrated to be crucial for cadherin (unction, the role of β-catenin is not yet fully understood. In this study, we analyzed the cadherin-catenin system in two human cell lines, HSC-39 and its putative subline HSC-40A, derived from a signet ring cell carcinoma of stomach. These cells grow as loose aggregates or single cells, suggesting that their cadherin system is not functional. In these cell lines, an identical 321-base pair in-frame mRNA deletion of β-catenin was identified; this led to a 107-amino-acid deletion in the NH2-terminal region of the protein. Southern blot analysis disclosed a homozygous deletion in part of the β-catenin gene. On the other hand, these cells expressed E-cadherin, α-catenin, and plakoglobin of normal size. Immunoprecipitation analyses showed that E-cadherin was coprecipitated with the mutated β-catenin but not with α-catenin, and antibodies against β-catenin did not copurify α-catenin. However, the recombinant fusion protein containing wild-type β-catenin precipitated α-catenin from these cells. These results suggest that the dysfunction of E-cadherin in these cell lines is due primarily to its failure to interact with α-catenin, and that this defect results from the mutation in β-catenin. Thus, it is most likely that the association between E-cadherin and α-catenin is mediated by β-catenin, and that this process is blocked by NH2-terminal deletion in β-catenin. These findings indicate that genetic abnormality of β-catenin is one of the mechanisms responsible for loosening of cell-cell contact, and may be involved in enhancement of tumor invasion in human cancers. © 1994, American Association for Cancer Research. All rights reserved.

E-cadherin gene mutations in human gastric carcinoma cell lines.

Oda T, Kanai Y, Oyama T, Yoshiura K, Shimoyama Y, Birchmeier W, Sugimura T, Hirohashi S.

Proc Natl Acad Sci USA (Proceedings of the National Academy of Sciences of the United States of America) 91 ( 5 ) 1858 - 1862 1994

Accepted, ISSN 00278424

Reduced expression of E-cadherin has been regarded as one of the main molecular events involved in dysfunction of the cell-cell adhesion system, triggering cancer invasion and metastasis. However, even with a sufficient amount of E-cadherin, cell-cell adhesion is sometimes lost in 'diffusely invasive' human carcinomas. Ten human cancer cell lines, showing growth characterized morphologically by loose cell-cell adhesion, were analyzed for possible structural abnormalities of their expressed E-cadherin. Four of the cell lines showed strong mRNA and protein expression with no nucleotide sequence abnormalities, and mRNA was absent in four other cell lines. mRNA sequence was abnormal in the remaining two gastric carcinoma cell lines. In MKN45 (poorly differentiated adenocarcinoma), this involved a 12-bp in-frame deletion with strong expression of mRNA and protein. In KATO-III (signet ring cell carcinoma), there were four mRNA species with insertions of different sizes, among which the major transcripts (with a 7-bp insertion) caused a frameshift, and expression of both mRNA and protein was markedly reduced. In these two cell lines, DNA mutations were detected around exon-intron junctions, revealing that aberrant RNA splicing was the cause of the mRNA abnormalities. In addition, the wild-type allele of the E-cadherin locus was lost, suggesting that the E-cadherin gene had been inactivated by two hits (mutation and allele loss), similar to the mechanism for inactivation of tumor suppressor genes.

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Abnormalities of cell adhesion molecules in multistage carcinogenesis

Kanai Y., Shimoyama Y., Oda T., Hirohashi S.

Gann Monographs on Cancer Research (Gann Monographs on Cancer Research) 42 127 - 138 1994

ISSN 00720151

Invasion and metastasis are later but critically important steps in multistage carcinogenesis. The inactivation of E (epithelial)-cadherin, which is a Ca2+-dependent cell-cell adhesion molecule, may trigger the release of cancer cells from the cancer nests. In fact, the invasion suppressing function of E-cadherin has been shown in in vitro studies. Furthermore, E-cadherin expression was strong in well differentiated carcinomas, whereas generally reduced in undifferentiated ones showing the strong invasive property. In spite of the above general rule, we found that a sizable proportion of undifferentiated gastric adenocarcinomas showing scattered histological growth pattern had definite E-cadherin expression. Therefore, human carcinoma cell lines lacking tight cell-cell adhesion were examined to clarify if alterations in catenins, which are cytoplasmic proteins interacting with cadherins, and/or structural abnormalities of normally expressed E-cadherin caused the dysfunction of the cell-cell adhesion system. Homologous deletion of part of the α-catenin gene with markedly reduced expression was observed in a human lung cancer cell line, PC9. mRNA sequence abnormalities of E-cadherin were detected in two human gastric cancer cell lines, MKN 45 and KATO-III. The wild-type allele of E-cadherin locus was lost in the two cell lines, indicating that E-cadherin gene was inactivated by a combination of loss of gene and mutation, similar to the mechanism which inactivated tumor suppressor genes. These findings suggested that varying genetic abnormalities of cell adhesion molecules could participate in multistage carcinogenesis, especially in the processes of invasion and metastasis.

Role of 5-hydroxytryptamine in the progression of monocrotaline induced pulmonary hypertension in rats.

Kanai Y, Hori S, Tanaka T, Yasuoka M, Watanabe K, Aikawa N, Hosoda Y.

Cardiovasc Res (Cardiovascular Research) 27 ( 9 ) 1619 - 1623 1993

Accepted, ISSN 00086363

Objective: The aim was to clarify the role of serotonin (5-hydroxytryptamine, 5-HT) in monocrotaline induced pulmonary hypertension. Methods: Plasma 5-HT levels, pulmonary capillary platelet count, and vascular responsiveness to 5-HT were evaluated in the model. The effects of the selective 5-HT2 receptor antagonist, DV- 7028, on the development of pulmonary hypertension were also investigated. Results: Plasma 5-HT was raised 12 h to 3 d after monocrotaline administration (60 mg·kg-1), coinciding with accumulation of platelets in the pulmonary circulation. Isolated pulmonary arteries showed hyperreactivity to 5-HT at 14 and 21 d after monocrotaline. Administration of DV-7028 (20 mg·kg-1·d-1) attenuated the increase in pulmonary arterial pressure, right ventricular hypertrophy, and medial thickening of the pulmonary arteries. Conclusions: The present study suggests that 5-HT released from platelets contributes to the initiation and progression of monocrotaline induced pulmonary hypertension.Cardiovascular Research 1993;27:1619-1623.

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Cloning of the human α-catenin cDNA and its aberrant mRNA in a human cancer cell line.

Oda T, Kanai Y, Shimoyama Y, Nagafuchi A, Tsukita S, Hirohashi S.

Biochem Biophys Res Commun (Biochemical and Biophysical Research Communications) 193 ( 3 ) 897 - 904 1993

Accepted, ISSN 0006291X

Cadherin and catenin compose cell adhesion complex and are indispensable for tight cell-cell adhesion. Dysfunction of this adhesion complex causes dissociation of cancer cells from primary tumor nodules, thus possibly contributing to cancer invasion and metastasis. In this report, we present the human α-caienin sequence. Human α-catenin showed extensive homology with that of mouse, i.e., 91.8% and 99.3% at the nucleic acid and amino acid levels, respectively, indicating that this molecule has been evolutionarily conserved in mammals. Characterization of the mRNA sequence of α-catenin in PC9 was also carried out, and two distinct abnormal sequences, i.e., one of 957 bp deletion resulting in a 319-amino-acid deletion and another of 761 bp deletion resulting in a frameshift, were identified. These deletions were probably produced by an error of RNA splicing, presenting one possible mechanism for the loss of intact α-catenin expression. © 1993 Academic Press, Inc.

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Pulmonary venoocclusive disease in a patient with systemic lupus erythematosus.

Kishida Y, Kanai Y, Kuramochi S, Hosoda Y.

J Rheumatol (Journal of Rheumatology) 20 ( 12 ) 2161 - 2162 1993

Accepted, ISSN 0315162X

Giant Cell Fibroblastoma: A Case Report and lmmunohistochemical Comparison with Ten Cases of Dermatofibrosarcoma Protuberans

Kanai Y., Mukai M., Sugiura H., Tanaka Y., Mukai K., Ukiyama E., Yokoyama J., Iri H., Hosoda Y.

Pathology International (Pathology International) 41 ( 7 ) 552 - 560 1991.07

ISSN 13205463

A 7 year old boy with giant cell fibroblastoma (GCF) of the skin and subcutaneous tissue of the right chest wall is described. To date, the histogenesis of GCF has not been clarified. The reason for the diversity of immunohistochemical data among various authors may be because the specimens studied were from only part of the lesion, or reduction of antigenicity through the preparation process. However, our findings based on studies of many specimens from various parts of the tumor for accurate immunohistochemical evaluation suggest that GCF may be a myofibro histiocytic tumor. Recently, the suggestion that GCF is a juvenile form of dermatofibrosarcoma protuberans (DFSP) has been reported. In addition to the present case, we performed immunohistochemical examination of 10cases of definitely diagnosed DFSP for comparison. The immunohistochemical characteristics of these two neoplasms were concordant. However, from clinical and morphological viewpoints, it seems premature to recognize GCF as a juvenile form of DFSP. Acta Pathol Jpn 41: 552–560, 1991. Copyright © 1991, Wiley Blackwell. All rights reserved

Access to Document (DOI)

Giant cell fibroblastoma. A case report and immunohistochemical comparison with ten cases of dermatofibrosarcoma protuberans.

Kanai Y, Mukai M, Sugiura H, Tanaka Y, Mukai K, Ukiyama E, Yokoyama J, Iri H, Hosoda Y.

Acta Pathol Jpn 41 552 - 560 1991

77-year-old woman with proteinuria: saddle nose and disorders in the central nervous system.

Saruta T, Sato M, Suzuki H, Akizuki M, Ushioda T, Hosoda Y, Yamawaki T, Kuramochi S, Sakaguchi H, Kanai Y.

Keio J Med 39 193 - 205 1990

Accepted

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