Division of Molecular Pathology

Yae Kanai, Eri Arai, Masahiro Gotoh, Ying Tian, Takuya Yotani, Koji Tsumura, Ayako Shibuya, Nanako Itoh

Introduction

On the basis of the findings from routine diagnostic pathology work, we develop scientific ideas and follow them up using a molecular pathological approach to understand the molecular basis of diseases and mechanisms determining clinicopathological heterogeneity of cancers. The Division of Molecular Pathology mainly consists of researchers who also belong to academia and industry. Therefore, we focus on the industry -academia-government collaboration to yield the potential benefits for cancer patients.

Research activities

1.Activities in the International Human Epigenome Consortium (IHEC)

We have participated in the IHEC as a principal investigator supported by the Core Research for Evolutional Science and Technology (CREST) project by the Japan Agency for Medical Research and Development (AMED). We have worked in collaboration with research groups in Kyushyu University and the University of Tokyo to reveal the epigenome landscape, whole-genome bisulfite sequencing using post-bisulfite adaptor tagging, chromatin immunoprecipitation sequencing, RNA sequencing, and whole-genome sequencing using normal epithelial cells purified from the liver, stomach, colon, and kidney.

In normal hepatocytes, CpG methylation levels were generally low in the region 200 bp upstream from the transcription start site (TSS200), the first coding exon and the CpG island. Considerable CHG and CHH methylation was observed. Personal differentially methylated regions (pDMRs) were observed less frequently in TSS200, the first coding exon and the CpG island. Histone modification profiles of pDMRs differed considerably among samples. pDMRs were observed around the TSSs of genes whose expression levels are reportedly regulated by CpG methylation. pDMRs were frequently observed in the vicinity of single-nucleotide variations and insertions/deletions, suggesting the possibility of cis-acting genome-epigenome interaction. Genetic variations may induce epigenetic variations and generate individual differences in the phenotypes of normal hepatocytes through variations in expression.

In 2016, we disclosed epigenome maps of normal purified hepatocytes and absorptive epithelial cells of the ascending colon, descending colon, and rectum from the National Bioscience Database Center (http://humandbs.biosciencedbc.jp/hum0032-v1) and the IHEC Data Portal (http://epigenomesportal.ca/ihec/index.html). Accurate epigenome profiling of normal cells will allow the identification of disease-specific epigenome profiles, thus facilitating a potential breakthrough in the prevention, diagnosis and therapy of diseases.

2.Epigenome analysis in human cancers

We analyzed single-institutional methylome data by single-CpG-resolution Infinium assay for samples of non-cancerous tissue and corresponding cancerous tissue obtained from the lung, stomach, kidney, breast, and liver. Gene ontology enrichment analysis showed that genes commonly methylated among cancers derived from various organs were enriched among "transcriptional factors" participating in the development and/or differentiation, which reportedly show bivalent histone modification in embryonic stem cells. Disruption of the differentiated state of precancerous cells via alterations of expression may be a common feature of DNA methylation alterations during carcinogenesis in multiple organs.

In order to clarify the significance of DNA methylation alterations during non-alcoholic steatohepatitis (NASH)-related hepatocarcinogenesis, which has shown an alarming increase in recent years, methylome analysis was performed. Significant DNA methylation alterations occurred in samples of non-cancerous liver tissue showing NASH (NASH-N) as compared with samples of normal liver tissue. Principal component analysis revealed distinct DNA methylation profiles of NASH samples that were different from those of samples of non-cancerous liver tissue showing chronic hepatitis or cirrhosis associated with hepatitis B or C virus infection. 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 NASH-related HCC themselves. NASH- and NASH-related HCC-specific DNA methylation alterations were observed in tumor-related genes, such as WHSC1, and were frequently associated with mRNA expression abnormalities.

In order to make DNA methylation diagnosis applicable to clinical use, a high performance liquid chromatography (HPLEC)-based and scaled-down methylated DNA detection device, which can be introduced into clinical laboratories of each hospital and even into small clinics, is now being developed by the joint research program with Sekisui Medical Co., Ltd. In 2016, national and international patents were established. We are now attempting to use this device for carcinogenetic risk estimation, liquid biopsy diagnosis, prognostication, and companion diagnosis for molecular targeted therapy in cancers derived from various organs.

3.Clinicopathological studies of human cancers based on the practice of diagnostic pathology

Using morphological, histological, immunohistochemical and molecular pathological approaches, diagnostic and prognostic criteria which are applicable to histological specimens, were explored. We collect tissue samples for the National Cancer Center Biobank and contribute to joint researches through providing clinicopathological information.

Future prospects

We will continuously perform joint research using tissue specimens pathologically examined by ourselves with researchers from both academia and industry to develop new strategies for cancer prevention, diagnosis, and therapy.

List of papers published in 2016

Journal

1.Ojima H, Yamagishi S, Shimada K, Shibata T. Establishment of various biliary tract carcinoma cell lines and xenograft models for appropriate preclinical studies. World J Gastroenterol, 22:9035-9038, 2016

2.Hiraoka N, Ino Y, Yamazaki-Itoh R. Tertiary Lymphoid Organs in Cancer Tissues. Front Immunol, 7:244, 2016

3.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. Mieap-regulated mitochondrial quality control is frequently inactivated in human colorectal cancer. Oncogenesis, 4:e181, 2016

4.Ohtomo-Oda R, Komatsu S, Mori T, Sekine S, Hirajima S, Yoshimoto S, Kanai Y, Otsuji E, Ikeda E, Tsuda H. SMYD2 overexpression is associated with tumor cell proliferation and a worse outcome in human papillomavirus-unrelated nonmultiple head and neck carcinomas. Hum Pathol, 49:145-155, 2016

5.Tanaka M, Nakajima T, Sugano K, Yoshida T, Taniguchi H, Kanemitsu Y, Nagino M, Sekine S. Mismatch repair deficiency in Lynch syndrome-associated colorectal adenomas is more prevalent in older patients. Histopathology, 69:322-328, 2016

6.Saito K, Arai E, Maekawa K, Ishikawa M, Fujimoto H, Taguchi R, Matsumoto K, Kanai Y, Saito Y. Lipidomic Signatures and Associated Transcriptomic Profiles of Clear Cell Renal Cell Carcinoma. Sci Rep, 6:28932, 2016

7.Morofuji N, Ojima H, Hiraoka N, Okusaka T, Esaki M, Nara S, Shimada K, Kishi Y, Kondo T. Antibody-based proteomics to identify an apoptosis signature for early recurrence of hepatocellular carcinoma. Clin Proteomics, 13:28, 2016

8.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. Macroscopic features predict outcome in patients with pancreatic ductal adenocarcinoma. Virchows Arch, 469:621-634, 2016

9.Sekine S, Yamashita S, Tanabe T, Hashimoto T, Yoshida H, Taniguchi H, Kojima M, Shinmura K, Saito Y, Hiraoka N, Ushijima T, Ochiai A. Frequent PTPRK-RSPO3 fusions and RNF43 mutations in colorectal traditional serrated adenoma. J Pathol, 239:133-138, 2016

10.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. 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. PLoS One, 11:e0167374, 2016