Glioma liquid biopsy of serum and cerebrospinal fluid using digital PCR and next-generation sequencer

• Project/Area Number: 17K10874
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Neurosurgery
• Research Institution: Kyorin University
• Principal Investigator: Saito Kuniaki 杏林大学, 医学部, 学内講師 (50446564)
• Project Period (FY): 2017-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000); Fiscal Year 2019: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2018: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000); Fiscal Year 2017: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: liquid biopsy / グリオーマ / digital PCR / cancer panel / glioma / 次世代シークエンサー / リキッドバイオプシー

Outline of Final Research Achievements

The aim of this research is to develop liquid biopsy for glioma using high-sensitive digital PCR. We extracted cell free DNA (cfDNA) from serum and cerebrospinal fluid and analyzed mutation and DNA methylation to achieve less-invasive diagnosis, early detection of recurrence and identification of biomarker. IDH1 mutation and TERT promoter mutation was identified with high sensitivity and specificity from cfDNA of glioma cases. Next generation sequencer (NGS) analysis of tumor DNA identified hypermutator phenotype and temporal heterogeneity of the glioma. Higher quality of tumor-derived circulating DNA was needed for liquid biopsy using NGS.

Academic Significance and Societal Importance of the Research Achievements

血清/髄液中のcfDNAを高感度解析することで、IDH変異やTERT変異などグリオーマの基本的な変異を検出することが高い感度特異度でできるようになった。グリオーマの非侵襲的な診断法の確立につながり、治療効果の判定や再発の検出としても画像診断より高い精度で行える可能性が示された。腫瘍の経時的な変化に伴う治療方針の最適化を行うことも可能となり、治療困難なグリオーマの予後改善に大きく貢献しうる。さらに、次世代シークエンサーを用いて経時的に解析が可能となれば、腫瘍の時間的多様性の解明から再発/悪性転化の機序の解明につながる可能性も秘めている。

Research Products

• [Journal Article] DNA demethylation is associated with malignant progression of lower-grade gliomas (2019)
  • Author(s): Nomura Masashi、Saito Kuniaki、Aihara Koki、Nagae Genta、Yamamoto Shogo、Tatsuno Kenji、Ueda Hiroki、Fukuda Shiro、Umeda Takayoshi、Tanaka Shota、…、Narita Yoshitaka、Nagane Motoo、Nishikawa Ryo、Ueki Keisuke、Saito Nobuhito、Aburatani Hiroyuki、Mukasa Akitake
  • Journal Title: Scientific Reports Volume: 9 Issue: 1 Pages: 1903-1903
  • DOI: 10.1038/s41598-019-38510-0
  • Peer Reviewed / Open Access
• [Presentation] グリオーマおよびグリオーマ幹細胞におけるIGFBP2メチル化解析 (2019)
  • Author(s): 5.齊藤邦昭、清水早紀、小林啓一、佐々木重嘉、山岸夢希、島田大輔、塩川芳昭、永根基雄
  • Organizer: 第20回日本分子脳神経外科学会
• [Presentation] がん遺伝子パネルにより同定した膠芽腫のhypermutator phenotype (2019)
  • Author(s): 9.齊藤邦昭、小林啓一、清水早紀、野崎江里子、佐々木重嘉、山岸夢希、島田大輔、飯島昌平、塩川芳昭、永根基雄
  • Organizer: 日本脳神経外科学会第78回学術総会
• [Presentation] Different types of hypermutator phenotype of glioblastoma according to mutation patterns of mismatch repair genes (2019)
  • Author(s): 11.Kuniaki Saito, Saki Shimizu, Eriko Nozaki, Keiichi Kobayashi, Nobuyoshi Sasaki, Yuki Yamagishi, Yoshiaki Shiokawa, Motoo Nagane
  • Organizer: 24th Annual meeting and education day of the Society for Neuro-Oncology
  • Int'l Joint Research
• [Presentation] Detailed analysis of mutation change after treatment in glioblastoma (2018)
  • Author(s): Kuniaki Saito, Saki Shimizu, Eriko Nozaki, Keiichi Kobayashi, Satoshi Kume, Tomohiro Chiba, Junji Shibahara, Yoshiaki Shiokawa, Motoo Nagane
  • Organizer: The 36th Annual Meeting of the Japan Society of Brain Tumor Pathology
  • Int'l Joint Research
• [Presentation] 膠芽腫に対するテモゾロミド治療の現状と薬剤耐性獲得の機序 (2018)
  • Author(s): 齊藤邦昭、清水早紀、野﨑江里子、小林啓一、久米賢、千葉知宏、柴原純二、塩川芳昭、永根基雄
  • Organizer: 日本脳神経外科学会第77回学術総会
• [Presentation] Mutation change after temozolomide treatment in primary glioblastoma (2018)
  • Author(s): Kuniaki Saito, Saki Shimizu, Eriko Nozaki, Keiichi Kobayashi, Yoshiaki Shiokawa, Motoo Nagane
  • Organizer: 第77回日本癌学会学術総会
• [Presentation] Detailed analysis of mutation change after treatment in glioblastoma (2018)
  • Author(s): Motoo Nagane, Kuniaki Saito, Saki Shimizu, Eriko Nozaki, Keiichi Kobayashi, Satoshi Kume, Tomohiro Chiba, Junji Shibahara, Yoshiaki Shiokawa,
  • Organizer: The 13th Meeting of the European Association of Neuro-Oncology
  • Int'l Joint Research
• [Presentation] Mechanism of acquired mutation after TMZ treatment (2018)
  • Author(s): Kuniaki Saito, Saki Shimizu, Eriko Nozaki, Keiichi Kobayashi, Satoshi Kume, Tomohiro Chiba, Junji Shibahara, Yoshiaki Shiokawa, Motoo Nagane
  • Organizer: 23rd Annual Meeting of the Society for Neuro-Oncology
  • Int'l Joint Research
• [Presentation] Prognostic significance of MGMT promoter and mismatch repair system in newly-diagnosed and recurrent malignant gliomas (2017)
  • Author(s): Kuniaki Saito, Saki Shimizu, Kaori Suzuki, Keiichi Kobayashi, Yoshiaki Shiokawa, Motoo Nagane
  • Organizer: 5th Quadrennial Meeting of the World Federation of Neuro-Oncology Societies (WFNOS)
  • Int'l Joint Research
• [Presentation] Integrated molecular analysis of newly diagnosed and recurrent glioblastoma after temozolomide treatment (2017)
  • Author(s): Kuniaki Saito, Kaori Suzuki, Saki Shimizu, Keiichi Kobayashi, Daisuke Shimada, Zentaro Kawaida, Tomohiro Chiba, Junji Shibahara, Yoshiaki Shiokawa, Motoo Nagane
  • Organizer: 第35回日本脳腫瘍病理学会
• [Presentation] Goal-setting of extent of resection on the basis of molecular status in glioma surgery (2017)
  • Author(s): Kuniaki Saito, Kaori Suzuki, Saki Shimizu, Keiichi Kobayashi, Daisuke Shimada, Zentaro Kawaida, Kazuhiro Chiba, Junji Shibahara, Yoshiaki Shiokawa, Motoo Nagane
  • Organizer: 第76回日本脳神経外科学会学術総会
• [Presentation] Temozolomide-induced mismatch repair insufficiency and hypermethylation of MGMT promoter with hypermutation in malignant glioma (2017)
  • Author(s): Kuniaki Saito, Saki Shimizu, Kaori Suzuki, Keiichi Kobayashi, Daisuke Shimada, Satosi Kume, Shohei Iijima, Tomohiro Chiba, Junji Shibahara, Yoshiaki Shiokawa, Motoo Nagane
  • Organizer: 22nd Annual Scientific Meeting and Education Day of the Society for Neuro-Oncology
  • Int'l Joint Research
• [Presentation] Integrated molecular analysis of newly diagnosed and recurrent glioblastoma after temozolomide treatment (2017)
  • Author(s): Kuniaki Saito, Saki Shimizu, Kaori Suzuki, Keiichi Kobayashi, Daisuke Shimada, Satosi Kume, Shohei Iijima, Tomohiro Chiba, Junji Shibahara, Yoshiaki Shiokawa, Motoo Nagane
  • Organizer: 第35回日本脳腫瘍学会学術集会