Establishment of a rapid intraoperative molecular analysis using the membrane patch method.

• Project/Area Number: 19K18728
• Research Category: Grant-in-Aid for Early-Career Scientists
• Allocation Type: Multi-year Fund
• Review Section: Basic Section 56050:Otorhinolaryngology-related
• Research Institution: Hamamatsu University School of Medicine
• Principal Investigator: Yamaguchi Yuki 浜松医科大学, 医学部附属病院, 診療助教 (60837821)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2021: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000); Fiscal Year 2020: ¥520,000 (Direct Cost: ¥400,000、Indirect Cost: ¥120,000); Fiscal Year 2019: ¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
• Keywords: メンブレンパッチ法 / リキッドバイオプシー / エピジェネティクス解析 / liquid biopsy / エピジェネティック解析

Outline of Research at the Start

切除後の迅速病理で断端に癌の残存を認めないにもかかわらず局所再発する、すなわち、顕微鏡的に確認できない癌や異形成の存在が疑われ、分子生物学的手法による切除断端、深部断端の癌残存有無の評価が必要である。
本申請は口腔癌切除後の深部断端における残存癌組織の有無を病理学的手法のみに頼らず、『メンブレンパッチ法』によるDNA採取、術前の組織生検(tissue biopsy)、採血からのDNAメチル化解析を併せ、より精度の高い診断法の確立と術中短時間DNAメチル化解析の実現を目標とし、“手術室からベンチそして手術室へ”とつなぐ「臨床へのリアルタイムな還元」を目指した斬新な研究である。

Outline of Final Research Achievements

In the first year, DNA was collected in three cases of tongue cancer using the membrane patch method; the average amount of DNA was 1.91 ug/ml on the tumor side and 1.19 ug/ml on the normal side. In the next year, the cytobrush method was introduced as an improvement to the membrane patch method. DNA was collected in 8 cases of oropharyngeal and tongue cancer cases. We were able to obtain DNA that were not different from the membrane patch method. In the final year, bisulfite treated DNA was analyzed the methylation status of E-cadherin, GALR1, TAC1, and COL1A2 was evaluated by Q-MSP method. Finally, we found that rapid molecular biological analysis by the cytobrush method was feasible within the same time frame as intraoperative pathology.

Academic Significance and Societal Importance of the Research Achievements

切除後の迅速病理で断端に癌の残存を認めないにもかかわらず局所再発する、すなわち、顕微鏡的に確認できない癌や異形成の存在が疑われ、分子生物学的手法による切除断端、深部断端の癌残存有無の評価が必要である。
本申請は中咽頭癌、舌癌症例の切除後の深部断端における残存癌組織の有無を病理学的手法のみに頼らず、『メンブレンパッチ法』によるDNA採取と『サイトブラシ法』によるDNA採取を比較し、術中短時間HPV解析とDNAメチル化解析の実現を施行し、分子生物学的迅速解析法の確立のための基礎でデーター蓄積を行い、臨床へのリアルタイムな還元の可能性を証明した。

Research Products

• [Journal Article] Prolonged activation of cytomegalovirus early gene e1-promoter exclusively in neurons during infection of the developing cerebrum (2021)
  • Author(s): Kosugi Isao、Arai Yoshifumi、Baba Satoshi、Kawasaki Hideya、Iwashita Toshihide、Tsutsui Yoshihiro
  • Journal Title: Acta Neuropathologica Communications Volume: 9 Issue: 8 Pages: 2165-2172
  • DOI: 10.7150/jca.54760
  • Peer Reviewed / Open Access
• [Journal Article] 頭頸部領域に発生したspindle cell carcinomaの8例 (2021)
  • Author(s): 山口 裕貴, 細川 誠二, 喜夛 淳哉, 望月 大極, 今井 篤志, 瀧澤 義徳, 三澤 清, 峯田 周幸
  • Journal Title: 耳鼻咽喉科臨床 補刷 Volume: 156 Pages: 61-65
  • NAID: 130007996850
  • Peer Reviewed / Open Access
• [Journal Article] Prostanoid receptor genes confer poor prognosis in head and neck squamous cell carcinoma via epigenetic inactivation. (2020)
  • Author(s): Misawa K, Mima M, Yamada S, Imai A, Mochizuki D, Ishikawa R, Kita J, Yamaguchi Y, Endo S, Misawa Y and Mineta H
  • Journal Title: Journal of Translational Medicine Volume: 18(1) Issue: 1 Pages: 31-31
  • DOI: 10.1186/s12967-020-02214-1
  • Peer Reviewed / Open Access
• [Journal Article] Neuropeptide receptor genes GHSR and NMUR1 are candidate epigenetic biomarkers and predictors for surgically treated patients with oropharyngeal cancer. (2020)
  • Author(s): Misawa K, Mima M, Yamada S, Misawa Y, Imai A, Mochizuki D, Nakagawa T, Kurokawa T, Oguro M, Ishikawa R, Yamaguchi Y, Endo S, Kawasaki H, Kanazawa Takeharu and Mineta H
  • Journal Title: Scientific Reports Volume: 10(1) Issue: 1 Pages: 1007-1007
  • DOI: 10.1038/s41598-020-57920-z
  • Peer Reviewed / Open Access
• [Journal Article] G protein-coupled receptor genes, PTGDR1, PTGDR2, and PTGIR, are candidate epigenetic biomarkers and predictors for treated patients with HPV-associated oropharyngeal cancer. (2020)
  • Author(s): Misawa K, Imai A, Kanazawa T, Mima M, Yamada S, Misawa Y, Mochizuki D, Yamada T, Shinmura D, Ishikawa R, Kita J, Yamaguchi Y, Misawa Y and Mineta H
  • Journal Title: Microorganisms Volume: 8(10) Issue: 10 Pages: 1504-1504
  • DOI: 10.3390/microorganisms8101504
  • Peer Reviewed / Open Access
• [Journal Article] Long interspersed nuclear element 1 hypomethylation has novel prognostic value and potential utility in liquid biopsy for oral cavity cancer. (2020)
  • Author(s): Misawa K, Yamada S, Mima M, Nakagawa T, Kurokawa T, Imai A, Mochizuki D, Shinmura D, Yamada T, Kita J, Ishikawa R, Yamaguchi Y, Misawa Y, Kanazawa T, Kawasaki H and Mineta H
  • Journal Title: Biomarker Research Volume: 8 Issue: 1 Pages: 53-53
  • DOI: 10.1186/s40364-020-00235-y
  • Peer Reviewed / Open Access