A new diagnostic algorithm using biopsy specimens in adult T-cell leukemia/lymphoma: combination of RNA in situ hybridization and quantitative PCR for HTLV-1

Research Project

Project/Area Number	19K07438
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Multi-year Fund
Section	一般
Review Section	Basic Section 49020:Human pathology-related
Research Institution	Nagoya University (2021) University of the Ryukyus (2019-2020)
Principal Investigator	Karube Kennosuke 名古屋大学, 医学系研究科, 教授 (20508577)
Project Period (FY)	2019-04-01 – 2022-03-31
Project Status	Completed (Fiscal Year 2021)
Budget Amount *help	¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000) Fiscal Year 2021: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000) Fiscal Year 2020: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000) Fiscal Year 2019: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Keywords	成人T細胞白血病/リンパ腫 / HTLV-1 / HBZ / RNAscope / in situ hybridization / FFPE標本 / tax
Outline of Research at the Start	ATLLの病変組織におけるHTLV-1ウイルスの同定法として、HBZ-in situ hybridization法を新しく開発する。この結果をHTLV-1ゲノム定量法、サザンブロット法などの従来より用いられている方法と比較しながら、その有効性および限界を把握し、日常診断において有用な検査にする。さらに、臨床経過や他のウイルス関連転写因子の発現との相関を解析しながら、ATLLの病態の解明に取り組む。
Outline of Final Research Achievements	Histopathological distinction between adult T-cell leukemia/lymphoma (ATLL) and other T-cell neoplasms is often challenging. Here we developed a new diagnostic algorithm for the accurate diagnosis of ATLL using FFPE samples. This method combines two HTLV-1 detection assays, namely, ultrasensitive RNA in situ hybridization using RNAscope for HTLV-1 bZIP factor (HBZ-RNAscope), and quantitative PCR targeting the tax gene (tax-qPCR). As a result, tax-qPCR had a higher ATLL identification rate than HBZ-RNAscope (88% [52/59], and 63% [39/62], respectively). However, HBZ-RNAscope clearly visualized the localization of HTLV-1-infected tumor cells and its identification rate increased to 94% (17/18) when the analysis was limited to samples up to 2 years old, indicating its usefulness in the daily diagnosis. This method is expected to replace Southern blot hybridization (SBH) and increase the accuracy of the diagnosis of ATLL.
Academic Significance and Societal Importance of the Research Achievements	ATLLの診断のためにはHTLV-1ウイルスの存在を証明する必要があるが、これまで手技が煩雑で生検体が必要なサザンブロット法が主流であった。今回の研究によりホルマリン固定標本でもウイルスの検出が可能になったことで、ATLLの病理学的診断における迅速性、正確性が大きく高まったと言える。さらに、病変組織中で直接ウイルスを「見る」ことができることで、ATLLの病態解明にも大きく貢献したと言える。