Study for novel biomarker in liquid biopsy on on carcinogenic pathways induced by driver mutations

• Project/Area Number: 20K07671
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 50020:Tumor diagnostics and therapeutics-related
• Research Institution: Center for Clinical and Biomedical Research, Sapporo Higashi Tokushukai Hospital
• Principal Investigator: Ono Yusuke 医療法人徳洲会札幌東徳洲会病院医学研究所, ゲノム診断研究部, 部門長 (40742648)
• Co-Investigator(Kenkyū-buntansha): 水上 裕輔 旭川医科大学, 医学部, 教授 (30400089) ; 唐崎 秀則 医療法人徳洲会札幌東徳洲会病院医学研究所, がん生物研究部, 客員研究員 (50374806)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2022: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2021: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2020: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: リキッドバイオプシー / ドライバー変異 / 遊離RNA / デジタルPCR / 膵癌 / IPMN / がん早期発見 / 遊離核酸 / バイオマーカー / 癌バイオマーカー / 早期発見

Outline of Research at the Start

リキッドバイオプシーは、がんのゲノム異常を俯瞰的にプロファイリングする技術として期待されるが、現状では早期癌の発見には好成績をあげられてはいない。我々は、そのなかでも特に早期発見が難しい膵癌において早くから遊離核酸中のドライバー遺伝子の高感度検出系の開発に取り組んできたが、DNA変異情報のみでの低い腫瘍検出率は課題とされた。
本研究では、我々が示した膵癌の前駆病変の一つである膵管内乳頭粘液性腫瘍(IPMN)における発癌プロセスに関する情報をもとに、ドライバー遺伝子と連動する特徴的なRNAプロファイルの探索を行い、IPMN患者血液中に存在する遊離RNAからの検出を目指す。

Outline of Final Research Achievements

In this study, we searched for new biomarkers targeting RNAs linked to driver genes using pancreatic cancer as a model. We generated KRAS mutant, GNAS mutant, KRAS/GNAS mutant, and wild-type strains of these genes in two different pancreatic ductal epithelial cell lines (HPDE, HPNE) and analyzed their expression patterns by RNA-seq. Furthermore, an effective method for collecting and purifying unstable plasma cell-free mRNA was validated, and a highly sensitive expression detection system using digital PCR was constructed. cfRNA was extracted from 35 PDA patient samples, and the amounts present were compared after quantification.

Academic Significance and Societal Importance of the Research Achievements

国内現行の血漿CGP検査は、現時点では進行癌患者を対象とし、検査目的は抗がん剤・分子標的薬の選択に限定される。研究ベースでは遺伝子変異にメチル化検出を組み合わせた包括的パネルが発表され、またmiRNAについて多癌腫のmiRNA検出パネルも検討されてきた。一方で本研究の対象としたmRNA、ncRNAはアポトーシス/壊死腫瘍細胞からの情報とシグナル伝達などの活性腫瘍細胞からの情報の両方、すなわち癌に関する直接のリアルタイム情報を取得できる可能性があり、本研究で目指した効果的な遊離RNAの回収と検出系を用いることにより、上記パネルと比較すると、特定の癌腫を早期で捉えるバイオマーカーになり得る。

Research Products

• [Journal Article] Multiplex Digital PCR Assay to Detect Multiple KRAS and GNAS Mutations Associated with Pancreatic Carcinogenesis from Minimal Specimen Amounts (2023)
  • Author(s): Maeda Chiho、Ono Yusuke、Hayashi Akihiro、Takahashi Kenji、Taniue Kenzui、Kakisaka Rika、Mori Miyuki、Ishii Takahiro、Sato Hiroki、Okada Tetsuhiro、Kawabata Hidemasa、Goto Takuma、Tamamura Nobue、Omori Yuko、Takahashi Kuniyuki、Katanuma Akio、Karasaki Hidenori、Liss Andrew Scott、Mizukami Yusuke
  • Journal Title: The Journal of Molecular Diagnostics Volume: 23 Issue: 6 Pages: 1525-1578
  • DOI: 10.1016/j.jmoldx.2023.02.007
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Molecular comparison of concurrent components of high-grade dysplasia, adenocarcinoma, and sarcomatoid carcinoma in a case of sarcomatoid carcinoma of the gallbladder (2023)
  • Author(s): Hirose K, Omori Y, Ono Y, Mizukami Y, Kaneko Y, Maruyama T, Ohtani H, Furukawa T.
  • Journal Title: Virchows Archiv Volume: 99 Issue: 2 Pages: 99-99
  • DOI: 10.1007/s00428-023-03524-7
  • Peer Reviewed
• [Journal Article] Local recurrence after successful endoscopic submucosal dissection for rectal mucinous mucosal adenocarcinoma: A case report (2023)
  • Author(s): Murakami Yuki、Tanabe Hiroki、Ono Yusuke、Sugiyama Yuya、Kobayashi Yu、Kunogi Takehito、Sasaki Takahiro、Takahashi Keitaro、Ando Katsuyoshi、Ueno Nobuhiro、Kashima Shin、Yuzawa Sayaka、Moriichi Kentaro、Mizukami Yusuke、Fujiya Mikihiro、Okumura Toshikatsu
  • Journal Title: World Journal of Gastrointestinal Oncology Volume: 15 Issue: 1 Pages: 186-194
  • DOI: 10.4251/wjgo.v15.i1.186
  • Peer Reviewed
• [Journal Article] GATA6 and CK5 Stratify the Survival of Patients With Pancreatic Cancer Undergoing Neoadjuvant Chemotherapy (2023)
  • Author(s): Kokumai T, Omori Y, Ishida M, Ohtsuka H, Mizuma M, Nakagawa K, Maeda C, Ono Y, Mizukami Y, Miura S, Kume K, Masamune A, Morikawa T, Unno M, Furukawa T.
  • Journal Title: Modern Pathology Volume: 36 Issue: 5 Pages: 100102-100102
  • DOI: 10.1016/j.modpat.2023.100102
  • Peer Reviewed
• [Journal Article] Distinct effects of TU-100 (daikenchuto) on long-lasting dysbiosis in the small intestine in patients with colorectal cancer and inflammatory bowel disease (2022)
  • Author(s): Kono T, Maejima T, Ono Y, Ito T, Furukawa S, Nishiyama M, Yamamoto M, Sugitani A, Karasaki H, Mizukami Y, Maemoto A.
  • Journal Title: Gene Volume: 820 Pages: 146266-146266
  • DOI: 10.1016/j.gene.2022.146266
  • Peer Reviewed
• [Journal Article] Mutant GNAS limits tumor aggressiveness in established pancreatic cancer via antagonizing the KRAS-pathway (2022)
  • Author(s): Kawabata Hidemasa、Ono Yusuke、Tamamura Nobue、Oyama Kyohei、Ueda Jun、Sato Hiroki、Takahashi Kenji、Taniue Kenzui、Okada Tetsuhiro、Fujibayashi Syugo、Hayashi Akihiro、ほか
  • Journal Title: Journal of Gastroenterology Volume: 57 Issue: 3 Pages: 208-220
  • DOI: 10.1007/s00535-021-01846-4
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Serine/Threonine Kinase 11 Plays a Canonical Role in Malignant Progression of KRAS-mutant and GNAS-wild-type Intraductal Papillary Mucinous Neoplasms of the Pancreas (2021)
  • Author(s): Omori Yuko、Ono Yusuke、Morikawa Takanori、Motoi Fuyuhiko、Higuchi Ryota、Yamamoto Masakazu、Hayakawa Yuko、Karasaki Hidenori、Mizukami Yusuke、Unno Michiaki、Furukawa Toru
  • Journal Title: Annals of Surgery Volume: Publish Ahead of Print Issue: 2 Pages: e384-e395
  • DOI: 10.1097/sla.0000000000004842
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Intracholecystic papillary neoplasm arising in the cystic duct and extending into common bile duct: a case report. (2021)
  • Author(s): Masahiro I, Masamichi M, Yasutaka A, Shuichi A, Tatsuo H, Tatsuyuki T, Kei K, Kunihiro M, Masaharu I, Hideo O, Kei N, Hiroki H, Takanori M, Takashi K, Kiyoshi K, Atsushi K, Atsushi M, Yuko O, Yusuke O, Yusuke M, Toru F, Michiaki U.
  • Journal Title: Clinical journal of gastroenterology Volume: 2 Issue: 2 Pages: 668-677
  • DOI: 10.1007/s12328-020-01311-4
  • Peer Reviewed
• [Journal Article] Prognostic significance of skeletal muscle decrease in unresectable pancreatic cancer: Survival analysis using the Weibull exponential distribution model (2021)
  • Author(s): Sato Hiroki、Goto Takuma、Hayashi Akihiro、Kawabata Hidemasa、Okada Tetsuhiro、Takauji Shuhei、Sasajima Junpei、Enomoto Katsuro、Fujiya Mikihiro、Oyama Kyohei、Ono Yusuke、Sugitani Ayumu、Mizukami Yusuke、Okumura Toshikatsu
  • Journal Title: Pancreatology Volume: 21 Issue: 5 Pages: 892-902
  • DOI: 10.1016/j.pan.2021.03.002
  • Peer Reviewed / Open Access
• [Journal Article] Monitoring epidermal growth factor receptor C797S mutation in Japanese non?small cell lung cancer patients with serial cell‐free DNA evaluation using digital droplet PCR (2021)
  • Author(s): Ariyasu Ryo、Uchibori Ken、Sasaki Takaaki、Tsukahara Mika、Kiyotani Kazuma、Yoshida Ryohei、Ono Yusuke、Kitazono Satoru、Ninomiya Hironori、Ishikawa Yuichi、Mizukami Yusuke、Yanagitani Noriko、Fujita Naoya、Nishio Makoto、Katayama Ryohei
  • Journal Title: Cancer Science Volume: - Issue: 6 Pages: 2371-2380
  • DOI: 10.1111/cas.14879
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] The feasibility of circulating tumor DNA analysis as a marker of recurrence in triple-negative breast cancer (2021)
  • Author(s): Okazaki Satoshi、Sasaki Takaaki、Yasuda Shunsuke、Abe Masahiro、Yoshida Nana、Yoshida Ryohei、Ishibashi Kei、Minami Yoshinori、Okumura Shunsuke、Chiba Shinichi、Takei Hidehiro、Hayashi Ryusuke、Nagato Toshihiro、Kobayashi Hiroya、Sugitani Ayumu、Ono Yusuke、Mizukami Yusuke、Kitada Masahiro、Ohsaki Yoshinobu
  • Journal Title: Oncology Letters Volume: 21 Issue: 5
  • DOI: 10.3892/ol.2021.12681
  • Peer Reviewed
• [Journal Article] Pathways for the development of multiple epithelial types of intraductal papillary mucinous neoplasm of the pancreas (2021)
  • Author(s): Kobayashi Toshikazu、Omori Yuko、Ono Yusuke、Karasaki Hidenori、Mizukami Yusuke、Makino Naohiko、Motoi Fuyuhiko、Unno Michiaki、Ueno Yoshiyuki、Furukawa Toru
  • Journal Title: Journal of Gastroenterology Volume: Online ahead of print Issue: 6 Pages: 581-592
  • DOI: 10.1007/s00535-021-01783-2
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] A Case of Adult Pancreatoblastoma With Novel APC Mutation and Genetic Heterogeneity (2021)
  • Author(s): Suemitsu Yamato、Ono Yusuke、Mizukami Yusuke、Ye Juanjuan、Yamakawa Keiko、Takamoto Takeshi、Nakano-Narusawa Yuko、Mukai Yuri、Takamatsu Manabu、Nakazawa Atsuko、Mino-Kenudson Mari、Kumasaka Toshio、Matsuda Yoko
  • Journal Title: Frontiers in Oncology Volume: 11 Pages: 99-99
  • DOI: 10.3389/fonc.2021.725290
  • Peer Reviewed
• [Journal Article] Case Report: A Rare Case of Esophagogastric Junctional Squamous Cell Carcinoma After the Successful Treatment of Neuroendocrine Carcinoma: Clonal Tumor Evolution Revealed by Genetic Analysis (2021)
  • Author(s): Sato Hiroki、Ono Yusuke、Mizukami Yusuke、Okumura Toshikatsu et al
  • Journal Title: Frontiers in Genetics Volume: 12 Pages: 608324-608324
  • DOI: 10.3389/fgene.2021.608324
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Long Non-Coding RNAs in Epithelial-Mesenchymal Transition of Pancreatic Cancer (2021)
  • Author(s): Takahashi Kenji、Taniue Kenzui、Ono Yusuke、Fujiya Mikihiro、Mizukami Yusuke、Okumura Toshikatsu
  • Journal Title: Frontiers in Molecular Biosciences Volume: 8 Pages: 717890-717890
  • DOI: 10.3389/fmolb.2021.717890
  • Peer Reviewed / Open Access
• [Journal Article] Time-saving method for directly amplifying and capturing a minimal amount of pancreatic tumor-derived mutations from fine-needle aspirates using digital PCR (2020)
  • Author(s): Yusuke Ono, Akihiro Hayashi, Chiho Maeda, Mayumi Suzuki, Reona Wada, Hiroki Sato, Hidemasa Kawabata, Tetsuhiro Okada, Takuma Goto, Hidenori Karasaki, Yusuke Mizukami, Toshikatsu Okumura
  • Journal Title: Scientific Reports Volume: 10 Issue: 1 Pages: 12332-12332
  • DOI: 10.1038/s41598-020-69221-6
  • Peer Reviewed / Open Access
• [Journal Article] Digital PCR-based plasma cell-free DNA mutation analysis for early-stage pancreatic tumor diagnosis and surveillance (2020)
  • Author(s): Okada T, Mizukami Y, Ono Y, Sato H, Hayashi A, Kawabata H, Koizumi K, Masuda S, Teshima S, Takahashi K, Katanuma A, Omori Y, Iwano H, Yamada M, Yokochi T, Asahara S, Kawakubo K, Kuwatani M, Sakamoto N, Enomoto K, Goto T, Sasajima J, Fujiya M, Ueda J, Matsumoto S, Taniue K, Sugitani A, Karasaki H, Okumura T
  • Journal Title: J Gastroenterol Volume: 55 Issue: 12 Pages: 1183-1193
  • DOI: 10.1007/s00535-020-01724-5
  • Peer Reviewed
• [Journal Article] How does intestinal-type intraductal papillary mucinous neoplasm emerge? CDX2 plays a critical role in the process of intestinal differentiation and progression. (2020)
  • Author(s): Omori Y, Ono Y, Kobayashi T, Motoi F, Karasaki H, Mizukami Y, Makino N, Ueno Y, Unno M, Furukawa T.
  • Journal Title: Virchows Archiv Volume: - Issue: 1 Pages: 1-11
  • DOI: 10.1007/s00428-020-02806-8
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Resection for pancreatic cancer metastases contributes to survival: A case report with sequential tumor genotype profiling during the long-term postoperative course (2020)
  • Author(s): Sato H, Sasajima J, Okada T, Hayashi A, Kawabata H, Goto T, Koizumi K, Tamamura N, Tanabe H, Fujiya M, Chiba S, Tanino M, Ono Y, Mizukami Y, Okumura T.
  • Journal Title: Medicine (Baltimore) Volume: 99 Issue: 25 Pages: e20564-e20564
  • DOI: 10.1097/md.0000000000020564
  • Peer Reviewed / Open Access
• [Journal Article] Genetic alteration of colorectal adenoma‐carcinoma sequence among gastric adenocarcinoma and dysplastic lesions in a patient with attenuated familial adenomatous polyposis (2020)
  • Author(s): Hiroki Tanabe, Kentaro Moriichi, Keitaro Takahashi, Yusuke Ono, Yu Kobayashi, Yuki Murakami, Takuya Iwama, Takehito Kunogi, Takahiro Sasaki, Katsuyoshi Ando, Nobuhiro Ueno, Shin Kashima, Hidehiro Takei, Yusuke Mizukami, Mikihiro Fujiya, Toshikatsu Okumura
  • Journal Title: Molecular Genetics & Genomic Medicine Volume: 8 Issue: 9
  • DOI: 10.1002/mgg3.1348
  • Peer Reviewed / Open Access
• [Journal Article] Serrated adenomas with a BRAF mutation in a young patient with familial adenomatous polyposis. (2020)
  • Author(s): Kentaro Moriichi, Hiroki Tanabe, Yusuke Ono, Yu Kobayashi, Yuki Murakami, Takuya Iwama, Takehito Kunogi, Takahiro Sasaki, Keitaro Takahashi, Katsuyoshi Ando, Nobuhiro Ueno, Shin Kashima, Hidehiro Takei, Yusuke Mizukami, Mikihiro Fujiya, Toshikatsu Okumura
  • Journal Title: International journal of colorectal disease Volume: 35 Issue: 10 Pages: 1967-1972
  • DOI: 10.1007/s00384-020-03657-0
  • Peer Reviewed
• [Journal Article] Intraductal papillary neoplasms of the bile duct consist of two distinct types specifically associated with clinicopathological features and molecular phenotypes (2020)
  • Author(s): Aoki Yasutaka、Mizuma Masamichi、Hata Tatsuo、Aoki Takeshi、Omori Yuko、Ono Yusuke、Mizukami Yusuke、Unno Michiaki、Furukawa Toru
  • Journal Title: The Journal of Pathology Volume: 251 Issue: 1 Pages: 38-48
  • DOI: 10.1002/path.5398
  • Peer Reviewed / Open Access
• [Journal Article] Genetic Tracing of Clonal Expansion and Progression of Pancreatic Ductal Adenocarcinoma: A Case Report and Multi-Region Sequencing Analysis (2020)
  • Author(s): Tachibana Shion、Mizukami Yusuke、Ono Yusuke、Sugiyama Yuya、Okada Tetsuhiro、Kitazaki Arisa、Sasajima Junpei、Tominaga Motoya、Sakamoto Jun、Kimura Keisuke、Omori Yuko、Furukawa Toru、Kimura Taichi、Tanaka Shinya、Nagashima Kazuo、Karasaki Hidenori、Ohta Tomoyuki、Okumura Toshikatsu
  • Journal Title: Frontiers in Oncology Volume: 10 Pages: 1-8
  • DOI: 10.3389/fonc.2020.00728
  • Peer Reviewed / Open Access
• [Journal Article] Metachronous intraductal papillary mucinous neoplasms disseminate via the pancreatic duct following resection (2019)
  • Author(s): Nagai Kazumasa、Mizukami Yusuke、Omori Yuko、Kin Toshifumi、Yane Kei、Takahashi Kuniyuki、Ono Yusuke、Sugitani Ayumu、Karasaki Hidenori、Shinohara Toshiya、Furukawa Toru、Hayashi Tsuyoshi、Okumura Toshikatsu、Maguchi Hiroyuki、Katanuma Akio
  • Journal Title: Modern Pathology Volume: 33 Issue: 5 Pages: 971-980
  • DOI: 10.1038/s41379-019-0405-7
  • Peer Reviewed / Open Access
• [Presentation] High-sensitive mutation detection in liquid biopsy with duodenal fluid captures genetic mutations associated with pancreatic carcinogenesis (2022)
  • Author(s): Yusuke Ono, Chiho Maeda, Kenji Takahashi, Hidenori Karasaki, Yusuke Mizukami
  • Organizer: AACR Special Conference; Pancreatic Cancer 2022; Plenary Session 5 "Prevention & Interception"
  • Int'l Joint Research
• [Presentation] Molecular barcode sequencing using duodenal fluid for profiling genomic alterations in pancreatic neoplasms (2022)
  • Author(s): Yusuke Ono, Kenji Takahashi, Hidenori Karasaki, Yusuke Mizukami
  • Organizer: Joint congress of the 26th meeting of international association of pancreatology and the 53rd annual meeting of japan pancreas society
  • Int'l Joint Research
• [Presentation] 消化液に存在する膵癌由来の遺伝子変異検出を目指したリキッドバイオプシー (2022)
  • Author(s): 小野裕介, 前田知歩, 高橋賢治, 河端秀賢, 唐崎秀則, 水上裕輔
  • Organizer: 第81回 日本癌学会 学術総会
• [Presentation] 分子バーコードを用いた 十二指腸液由来核酸の変異解析 -より高濃度の変異検出をターゲットとした 膵癌リキッドバイオプシー- (2022)
  • Author(s): 小野 裕介、前田 知歩、高橋 賢治、唐崎 秀則、水上 裕輔
  • Organizer: 第6回 Liquid Biopsy研究会
• [Presentation] 分子バーコードを用いた十二指腸液由来核酸の変異解析 -より高濃度の変異検出をターゲットとした膵癌リキッドバイオプシー-- (2021)
  • Author(s): 小野 裕介、前田 知歩、高橋 賢治、唐崎 秀則、水上 裕輔
  • Organizer: 第80回 日本癌学会 学術総会
• [Presentation] 臨床残余検体を用いた遺伝子変異検出のためのマルチプレックスデジタルPCR前増幅法の確立 (2021)
  • Author(s): 前田 知歩、小野 裕介、高橋 賢治、水上 裕輔
  • Organizer: 第80回 日本癌学会 学術総会
• [Presentation] Multiplex digital PCR for detection KRAS and GNAS mutations.マルチプレックスデジタルPCR法を用いたKRASおよびGNAS遺伝子変異解析 (2020)
  • Author(s): 前田知歩, 小野裕介, 水上裕輔
  • Organizer: 第79回日本癌学会学術総会
• [Book] がんゲノム病理学 (2021)
  • Author(s): 田中伸哉、西原広史 （小野担当:分担執筆, 範囲:第3章 ゲノムにおける解析手法 V.リキッドバイオプシー）
  • Total Pages: 234
  • Publisher: 文光堂
  • ISBN: 9784830604850
• [Remarks] 医療法人徳洲会 札幌東徳洲会病院 医学研究所HP
  • URL: https://www.higashi-tokushukai.or.jp/inst_biomed_res/
• [Patent(Industrial Property Rights)] 遺伝子解析用サンプルの製造方法 (2020)
  • Inventor(s): 水上裕輔、小野裕介、林明宏
  • Industrial Property Rights Holder: 水上裕輔、小野裕介、林明宏
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2020-217083
  • Filing Date: 2020
• [Patent(Industrial Property Rights)] 遺伝子解析用サンプルの製造方法 (2020)
  • Inventor(s): 水上裕輔、小野裕介、林明宏
  • Industrial Property Rights Holder: 水上裕輔、小野裕介、林明宏
  • Industrial Property Rights Type: 特許
  • Filing Date: 2020