Annual Report 2018

Department of Clinical Genomics

Hitoshi Ichikawa, Hiromi Sakamoto, Masahiro Gotoh, Kazuhiko Aoyagi, Takashi Kubo, Misuzu Tsukamoto, Yoko Odaka, Mineko Ushiama, Sachiyo Mitani, Fumito Yamazaki, Masumi Shimizu, Noriko Abe, Sayaka Mito, Erika Arakawa, Tomoko Ikegami, Akiko Sakamoto, Yuka Nishina, Emi Shigino, Tomoyo Oda

Introduction

 The aim of the Department of Clinical Genomics is to contribute to realize genome medicine for cancer patients through the development and implementation of nextgeneration sequencing (NGS)-based genomic tests and the identification of novel biomarkers and therapeutic targets from NGS analyses of cancer genomes. We have been developing two types of NGS-based panel tests: the germline test for hereditary cancer syndromes and the somatic tests for therapy selection, diagnosis, and prognostic prediction. In addition, we are providing genomic analysis services as a part of core facility functions of the Fundamental Innovative Oncology Core Center (FIOC) of the National Cancer Center Research Institute (NCCRI).

Research activities

1. Development of the germline NGS panel test for hereditary cancer syndromes

 We developed the NCC Oncopanel FC (Familial Cancer) test, an NGS panel test which can analyze 147 hereditary cancer syndromeassociated genes. This test shortened turnaround times substantially by simultaneous sequencing of target genes as compared to the previous capillary sequencing tests of each single gene one by one. Germline clinical testing using this system was performed for patients and their relatives visiting the Outpatient Genetic Counseling Clinic in the NCC-Hospital (NCCH) or other collaborating hospitals. A total of 264 patients were analyzed from April 2018 to March 2019. Detected variants were annotated for clinical significance by researchers of our department and physicians of the Department of Genetic Medicine and Services in the NCCH. At present, a number of variants remained as "variants of uncertain significance (VUS)". Data and knowledge accumulation would be crucial for the germline clinical sequencing.

2. Development of the somatic NGS panel tests for therapy selection, diagnosis, and prognostic prediction

 We developed the NCC Oncopanel test, an NGS panel test which can detect mutations and amplifications of 114 genes and fusions of 12 genes from FFPE tumor tissue samples. In December 2018, this test was approved by PMDA as a clinical test to detect actionable genetic alterations for therapy selection of cancer patients, based on the results of the TOP-GEAR study. In addition, we also developed a novel NGS panel test for pediatric cancers, NCC Oncopanel Ped, and started its analytical validation. This test aims for molecular diagnosis and risk stratification in addition to therapy selection of pediatric cancers, and can detect mutations and amplifications of 211 genes and fusions of nine genes.

3. Identification of novel biomarkers and therapeutic targets

 Through the NGS analysis of cancer genomes, we are searching for novel biomarkers and therapeutic targets for sarcoma and pediatric cancer. From April 2018 to March 2019, we conducted comprehensive genomic profiling of dedifferentiated liposarcoma (DDLPS) in collaboration with the Japan Sarcoma Genome Consortium (JSGC) and genomic characterization of adolescent and young adult (AYA) sarcoma. In addition, we also analyzed granular cell tumors (GCTs), rare mesenchymal tumors that exhibit a characteristic morphology and a finely granular cytoplasm. We performed wholeexome sequencing, RNA sequencing, and targeted sequencing of 51 GCT samples, and identified mutations in genes encoding vacuolar H+ -ATPase (V-ATPase) components, including ATP6AP1 and ATP6AP2, in 33 (65%) GCTs. ATP6AP1 and ATP6AP2 mutations were found in 23 (45%) and 2 (4%) samples, respectively, and all were truncating or splice site mutations. In addition, seven other genes encoding V-ATPase components were also mutated, and three mutations in ATP6V0C occurred on the same amino acid (isoleucine 136). These V-ATPase component gene mutations were mutually exclusive, with one exception. These results suggest that V-ATPase function is impaired in GCTs not only by loss-of-function mutations of ATP6AP1 and ATP6AP2 but also through mutations of other subunits, and that V-ATPase dysfunction promotes GCT tumorigenesis.

4. Core facility genomic analysis services

 We provided NGS and microarray analysis services as a part of core facility functions of the FIOC of the NCCRI. Upon requests from researchers not only in the NCCRI but also in the NCCH and the NCC-Hospital East (NCCHE), whole exome sequencing of 132 samples, targeted sequencing of 262 samples, RNA sequencing of five samples, and SNP array analysis of 1,568 samples were performed from April 2018 to March 2019.

Education

 The Department of Clinical Genomics accepted two undergraduate and one graduate students as trainees.

List of papers published in 2018

Journal

 1. Asano N, Matsuzaki J, Ichikawa M, Kawauchi J, Takizawa S, Aoki Y, Sakamoto H, Yoshida A, Kobayashi E, Tanzawa Y, Nakayama R, Morioka H, Matsumoto M, Nakamura M, Kondo T, Kato K, Tsuchiya N, Kawai A, Ochiya T. A serum microRNA classifier for the diagnosis of sarcomas of various histological subtypes. Nat Commun, 10:1299, 2019

 2. Ohki K, Kiyokawa N, Saito Y, Hirabayashi S, Nakabayashi K, Ichikawa H, Momozawa Y, Okamura K, Yoshimi A, Ogata-Kawata H, Sakamoto H, Kato M, Fukushima K, Hasegawa D, Fukushima H, Imai M, Kajiwara R, Koike T, Komori I, Matsui A, Mori M, Moriwaki K, Noguchi Y, Park MJ, Ueda T, Yamamoto S, Matsuda K, Yoshida T, Matsumoto K, Hata K, Kubo M, Matsubara Y, Takahashi H, Fukushima T, Hayashi Y, Koh K, Manabe A, Ohara A. Clinical and molecular characteristics of MEF2D fusion-positive B-cell precursor acute lymphoblastic leukemia in childhood, including a novel translocation resulting in MEF2D-HNRNPH1 gene fusion. Haematologica, 104:128-137, 2019

 3. Shiino S, Matsuzaki J, Shimomura A, Kawauchi J, Takizawa S, Sakamoto H, Aoki Y, Yoshida M, Tamura K, Kato K, Kinoshita T, Kitagawa Y, Ochiya T. Serum miRNA-based Prediction of Axillary Lymph Node Metastasis in Breast Cancer. Clin Cancer Res, 25:1817-1827, 2019

 4. Makise N, Sekimizu M, Konishi E, Motoi T, Kubo T, Ikoma H, Watanabe SI, Okuma T, Hiraoka N, Fukayama M, Kawai A, Ichikawa H, Yoshida A. H3K27me3 deficiency defines a subset of dedifferentiated chondrosarcomas with characteristic clinicopathological features. Mod Pathol, 32:435-445, 2019

 5. Tanikawa C, Kamatani Y, Toyoshima O, Sakamoto H, Ito H, Takahashi A, Momozawa Y, Hirata M, Fuse N, Takai-Igarashi T, Shimizu A, Sasaki M, Yamaji T, Sawada N, Iwasaki M, Tsugane S, Naito M, Hishida A, Wakai K, Furusyo N, Murakami Y, Nakamura Y, Imoto I, Inazawa J, Oze I, Sato N, Tanioka F, Sugimura H, Hirose H, Yoshida T, Matsuo K, Kubo M, Matsuda K. Genome-wide association study identifies gastric cancer susceptibility loci at 12q2411-12 and 20q1121. Cancer Sci, 109:4015-4024, 2018

 6. Sato Y, Ueno H, Ioka T, Ohkawa S, Ikeda M, Shimamura T, Tsuji A, Tsuchiya Y, Furuse J, Ishii H, Furuya K, Iguchi H, Saito Y, Kaniwa N, Sawada JI, Sakamoto H, Sekine A, Okusaka T, Yoshida T. SLCO1B1 Polymorphism Is a Drug Response Predictive Marker for Advanced Pancreatic Cancer Patients Treated With Gemcitabine, S-1, or Gemcitabine Plus S-1. Pancreas, 47:637-642, 2018

 7. Yokoi A, Matsuzaki J, Yamamoto Y, Yoneoka Y, Takahashi K, Shimizu H, Uehara T, Ishikawa M, Ikeda SI, Sonoda T, Kawauchi J, Takizawa S, Aoki Y, Niida S, Sakamoto H, Kato K, Kato T, Ochiya T. Integrated extracellular microRNA profiling for ovarian cancer screening. Nat Commun, 9:4319, 2018

 8. Toki S, Wakai S, Sekimizu M, Mori T, Ichikawa H, Kawai A, Yoshida A. PAX7 immunohistochemical evaluation of Ewing sarcoma and other small round cell tumours. Histopathology, 73:645-652, 2018

 9. Makise N, Sekimizu M, Kubo T, Wakai S, Hiraoka N, Komiyama M, Fukayama M, Kawai A, Ichikawa H, Yoshida A. Clarifying the Distinction Between Malignant Peripheral Nerve Sheath Tumor and Dedifferentiated Liposarcoma: A Critical Reappraisal of the Diagnostic Utility of MDM2 and H3K27me3 Status. Am J Surg Pathol, 42:656-664, 2018

10. Yotani T, Yamada Y, Arai E, Tian Y, Gotoh M, Komiyama M, Fujimoto H, Sakamoto M, Kanai Y. Novel method for DNA methylation analysis using high-performance liquid chromatography and its clinical application. Cancer Sci, 109:1690-1700, 2018

11. Makise N, Sekimizu M, Kubo T, Wakai S, Watanabe SI, Kato T, Kinoshita T, Hiraoka N, Fukayama M, Kawai A, Ichikawa H, Yoshida A. Extraskeletal osteosarcoma: MDM2 and H3K27me3 analysis of 19 cases suggest disease heterogeneity. Histopathology, 73:147-156, 2018

12. Fukuoka K, Kanemura Y, Shofuda T, Fukushima S, Yamashita S, Narushima D, Kato M, Honda-Kitahara M, Ichikawa H, Kohno T, Sasaki A, Hirato J, Hirose T, Komori T, Satomi K, Yoshida A, Yamasaki K, Nakano Y, Takada A, Nakamura T, Takami H, Matsushita Y, Suzuki T, Nakamura H, Makino K, Sonoda Y, Saito R, Tominaga T, Matsusaka Y, Kobayashi K, Nagane M, Furuta T, Nakada M, Narita Y, Hirose Y, Ohba S, Wada A, Shimizu K, Kurozumi K, Date I, Fukai J, Miyairi Y, Kagawa N, Kawamura A, Yoshida M, Nishida N, Wataya T, Yamaoka M, Tsuyuguchi N, Uda T, Takahashi M, Nakano Y, Akai T, Izumoto S, Nonaka M, Yoshifuji K, Kodama Y, Mano M, Ozawa T, Ramaswamy V, Taylor MD, Ushijima T, Shibui S, Yamasaki M, Arai H, Sakamoto H, Nishikawa R, Ichimura K. Significance of molecular classification of ependymomas: C11orf95-RELA fusion-negative supratentorial ependymomas are a heterogeneous group of tumors. Acta Neuropathol Commun, 6:134, 2018

13. Itahashi K, Kondo S, Kubo T, Fujiwara Y, Kato M, Ichikawa H, Koyama T, Tokumasu R, Xu J, Huettner CS, Michelini VV, Parida L, Kohno T, Yamamoto N. Evaluating Clinical Genome Sequence Analysis by Watson for Genomics. Front Med, 5:305, 2018

14. 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. Epigenome mapping of human normal purified hepatocytes: personal epigenome variation and genome-epigenome correlation. Epigenomics, 10:955- 979, 2018

15. Kato M, Nakamura H, Nagai M, Kubo T, Elzawahry A, Totoki Y, Tanabe Y, Furukawa E, Miyamoto J, Sakamoto H, Matsumoto S, Sunami K, Arai Y, Suzuki Y, Yoshida T, Tsuchihara K, Tamura K, Yamamoto N, Ichikawa H, Kohno T, Shibata T. A computational tool to detect DNA alterations tailored to formalin-fixed paraffin-embedded samples in cancer clinical sequencing. Genome Med, 10:44, 2018