Molecular Analysis of Liquid-Based Cytological Specimen Using Virtually Positive Sputum with Adenocarcinoma Cells
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...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-02-01
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| Series: | Diagnostics |
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| Online Access: | https://www.mdpi.com/2075-4418/10/2/84 |
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| author | Takeshi Nishikawa Tomomi Fujii Shigenobu Tatsumi Aya Sugimoto Yoko Sekita-Hatakeyama Keiji Shimada Masaharu Yamazaki Kinta Hatakeyama Chiho Ohbayashi |
| author_facet | Takeshi Nishikawa Tomomi Fujii Shigenobu Tatsumi Aya Sugimoto Yoko Sekita-Hatakeyama Keiji Shimada Masaharu Yamazaki Kinta Hatakeyama Chiho Ohbayashi |
| author_sort | Takeshi Nishikawa |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T08:06:02Z |
| format | Article |
| id | doaj.art-96c35ad9ffa9483aadf043ee020f8c1e |
| institution | Directory Open Access Journal |
| issn | 2075-4418 |
| language | English |
| last_indexed | 2024-04-13T08:06:02Z |
| publishDate | 2020-02-01 |
| publisher | MDPI AG |
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| series | Diagnostics |
| spelling | doaj.art-96c35ad9ffa9483aadf043ee020f8c1e2022-12-22T02:55:09ZengMDPI AGDiagnostics2075-44182020-02-011028410.3390/diagnostics10020084diagnostics10020084Molecular Analysis of Liquid-Based Cytological Specimen Using Virtually Positive Sputum with Adenocarcinoma CellsTakeshi Nishikawa0Tomomi Fujii1Shigenobu Tatsumi2Aya Sugimoto3Yoko Sekita-Hatakeyama4Keiji Shimada5Masaharu Yamazaki6Kinta Hatakeyama7Chiho Ohbayashi8Department of Diagnostic Pathology, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8521, JapanDepartment of Diagnostic Pathology, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8521, JapanDepartment of Diagnostic Pathology, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8521, JapanDepartment of Diagnostic Pathology, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8521, JapanDepartment of Diagnostic Pathology, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8521, JapanDepartment of Diagnostic Pathology, Nara City Hospital, Nara 630-8305, JapanDepartment of Central Clinical Laboratory, Nara Medical University Hospital, Nara 634-8521, JapanDepartment of Diagnostic Pathology, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8521, JapanDepartment of Diagnostic Pathology, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8521, JapanLiquid-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.https://www.mdpi.com/2075-4418/10/2/84lung adenocarcinomaegfr mutationliquid-based cytologyquantitative pcr |
| spellingShingle | Takeshi Nishikawa Tomomi Fujii Shigenobu Tatsumi Aya Sugimoto Yoko Sekita-Hatakeyama Keiji Shimada Masaharu Yamazaki Kinta Hatakeyama Chiho Ohbayashi Molecular Analysis of Liquid-Based Cytological Specimen Using Virtually Positive Sputum with Adenocarcinoma Cells Diagnostics lung adenocarcinoma egfr mutation liquid-based cytology quantitative pcr |
| title | Molecular Analysis of Liquid-Based Cytological Specimen Using Virtually Positive Sputum with Adenocarcinoma Cells |
| title_full | Molecular Analysis of Liquid-Based Cytological Specimen Using Virtually Positive Sputum with Adenocarcinoma Cells |
| title_fullStr | Molecular Analysis of Liquid-Based Cytological Specimen Using Virtually Positive Sputum with Adenocarcinoma Cells |
| title_full_unstemmed | Molecular Analysis of Liquid-Based Cytological Specimen Using Virtually Positive Sputum with Adenocarcinoma Cells |
| title_short | Molecular Analysis of Liquid-Based Cytological Specimen Using Virtually Positive Sputum with Adenocarcinoma Cells |
| title_sort | molecular analysis of liquid based cytological specimen using virtually positive sputum with adenocarcinoma cells |
| topic | lung adenocarcinoma egfr mutation liquid-based cytology quantitative pcr |
| url | https://www.mdpi.com/2075-4418/10/2/84 |
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