Quantification of fusion cfDNA and resistant mutation cfRNA in plasma EVs from patients with lung cancer

• Project/Area Number: 19K16847
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 50020:Tumor diagnostics and therapeutics-related
• Research Institution: Jikei University School of Medicine
• Principal Investigator: Yoshtiaka Seki 東京慈恵会医科大学, 医学部, 講師 (00733213)
• Project Period (FY): 2019-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2020: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2019: ¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000)
• Keywords: 治療耐性因子探索 / 融合遺伝子陽性肺がん / cfDNA / チロシンキナーゼ阻害薬 / 融合遺伝子陽性肺癌 / チロシンキナーゼ阻害剤 / リキッドバイオプシー / エクソソーム / 血中遊離核酸 / 遺伝子融合 / 個別化医療

Outline of Research at the Start

当グループでは血漿中のcell-free DNA(cfDNA)を用いた肺がん細胞の遺伝子変異・融合遺伝子の解析によるゲノムDNA上の変異・融合好発部位を報告している。この解析技術と情報を応用し、ゲノムDNAからの検出が困難とされているEML4-ALK融合遺伝子変異につき、次世代シークエンサーを用いた血漿中のcell-free DNA(cfDNA)解析での定量的解析を試みる。
さらに検証が終了した段階でEML4-ALK遺伝子融合陽性の肺腺がん患者の血漿検体を用いて、確立したシークエンシングを行うことにより、血漿からの融合変異/耐性変異の同定・定量と耐性機構判定法の確立を目指す。

Outline of Final Research Achievements

At first, we attempted to establish a method for quantitative detection of mutations by cfDNA and detection of fusion genes. As a result, we found that Guardant Health's commercial-based analysis method was useful for detecting fusion genes. Therefore, we decided to proceed with the estimation of resistance mechanisms from other related genes based on the company's analysis method, using a research protocol approved by the hospital research ethics review committee. A genetic search for resistance mechanisms was successfully conducted after collecting and analyzing blood samples from fusion gene-positive patients at the time of TKI resistance. As a result, he reported at the conference that candidate genes for fusion genes and resistance can be detected in blood samples, and that further analysis could lead to clinical applications.

Academic Significance and Societal Importance of the Research Achievements

fDNAによる変異の定量的検出、融合遺伝子の検出法の確立を試みた。その結果、融合遺伝子の検出についてはGuardant Health社のコマーシャルベースの解析法が有用である事が判明した。融合遺伝子陽性患者TKI耐性化時の血液サンプルを収集し解析した上で耐性機序の遺伝学的検索に成功した。融合遺伝子・耐性の候補遺伝子が血液検体から検出できること、今後のさらなる解析の積み重ねにより臨床応用も可能となりうることについて学会で報告した。

Research Products

• [Journal Article] Liquid biopsy for the detection of resistance mutations to ROS1 and RET inhibitors in non-small lung cancers: A case series study (2022)
  • Author(s): Seki Yoshitaka、Yoshida Tatsuya、Kohno Takashi、Masuda Ken、Okuma Yusuke、Goto Yasushi、Horinouchi Hidehito、Yamamoto Noboru、Kuwano Kazuyoshi、Ohe Yuichiro
  • Journal Title: Respiratory Investigation Volume: 60 Issue: 6 Pages: 852-856
  • DOI: 10.1016/j.resinv.2022.08.002
  • Peer Reviewed
• [Journal Article] P37-1 Liquid biopsy for the detection of the resistant mutation to ROS1 and RET inhibitors: A case series study (2022)
  • Author(s): Seki Yoshitaka, Yoshida Tatsuya, Masuda Ken, Horinouchi Hidehito, Shinno Yuki, Okuma Yusuke, Goto Yasushi, Yamamoto Noboru, Kuwano Kazuyoshi, Takashi Kohno, Ohe Yuichiro
  • Journal Title: Annals of Oncology Volume: 33 Pages: S528-S528
  • DOI: 10.1016/j.annonc.2022.05.292
• [Presentation] P37-1 Liquid biopsy for the detection of the resistant mutation to ROS1 and RET inhibitors: A case series study (2022)
  • Author(s): Seki Yoshitaka, Yoshida Tatsuya, Masuda Ken, Horinouchi Hidehito, Shinno Yuki, Okuma Yusuke, Goto Yasushi, Yamamoto Noboru, Kuwano Kazuyoshi, Takashi Kohno, Ohe Yuichiro
  • Organizer: 2022 the Japanese Society of Medical Oncology Annual Meeting, Kyoto, Japan
  • Int'l Joint Research
• [Presentation] Liquid biopsy for the detection of the resistant mutation to ROS1 and RET inhibitors: A case series study (2022)
  • Author(s): 関 好孝
  • Organizer: 第19回日本臨床腫瘍学会学術集会