Alternative Title液状化細胞診材料を用いた遺伝子解析による腫瘍特異的遺伝子検出感度の検討
Note (General)type:Thesis
Liquid-based cytology (LBC) analysis of sputum is a useful diagnostic and prognostic tool for detecting lung cancer. DNA and RNA derived from lung cancer cells can be used for this diagnosis. However, the quality of cytological material is not always adequate for molecular analysis due to the effect of formalin in the commercially available fixation kits. In this study, we examined DNA and RNA extraction methods for LBC analysis with formalin fixation, using lung carcinoma cell lines and sputum. The human non-small cell lung cancer cell lines were fixed with LBC fixation reagents, such as CytoRich red preservative. Quantification of thyroid transcription factor-1 (TTF-1) and actin mRNA, epidermal growth factor receptor (EGFR) DNA in HCC827, H1975, and H1299 cells, and mutation analysis of EGFR in HCC827 and H1975 cells were performed by quantitative PCR (qPCR) and fluorescence resonance energy transfer (FRET)-based preferential homoduplex formation assay (F-PHFA) method, respectively. mRNA and DNA extracted from cell lines using RNA and/or DNA extraction kits for formalin-fixed paraffin-embedded (FFPE) fixed with various LBC solutions were efficiently detected by qPCR. The detection limit of EGFR mutations was at a rate of 5% mutated positive cells in LBC. The detection limit of the EGFR exon 19 deletion in HCC827 was detected in more than 1.5% of the positive cells in sputum. In contrast, the detection limit of the T790M/L858R mutation in H1975 was detected in more than 13% of the positive cells. We also detected EGFR mutations using next generation sequencing (NGS). The detection limit of NGS for EGFR mutation was lower than that of the F-PHFA method. Furthermore, more than 0.1% of positive cells could be cytomorphologically detected. Our results demonstrate that LBC systems are powerful tools for cytopathological and genetic analyses. However, careful attention should be paid to the incidence of false negative results in the genetic analysis of EGFR mutations detected by LBC.
博士（医学）・甲第750号・令和2年6月30日
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
identifier:Diagnostics Vol.10 No.2 Article No.84 (2020 Feb)
identifier:20754418
identifier:http://ginmu.naramed-u.ac.jp/dspace/handle/10564/3757
identifier:Diagnostics Vol. No., 10(2): Article No.84
DOIinfo:doi/10.3390/diagnostics10020084
Collection (particular)国立国会図書館デジタルコレクション > デジタル化資料 > 博士論文
Date Accepted (W3CDTF)2020-11-10T19:58:22+09:00
Data Provider (Database)国立国会図書館 : 国立国会図書館デジタルコレクション