Abstract
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.
Highlights
Lung cancer is the leading cause of cancer deaths in the world with more than 80% of lung cancer cases being non-small cell lung carcinoma (NSCLC)
These results suggest that false-negative results may be observed in less than 1% of cancer cells in the detection of epidermal growth factor receptor (EGFR) mutation using the Liquid-based cytology (LBC) specimen
Molecular testing for EGFR mutations is one of the most important tools currently being used for the diagnosis and therapeutic management of NSCLC [21,22]
Summary
Lung cancer is the leading cause of cancer deaths in the world with more than 80% of lung cancer cases being non-small cell lung carcinoma (NSCLC). Molecular targeted therapy and immunotherapy are selected in addition to conventional chemotherapy for the treatment of Diagnostics 2020, 10, 84; doi:10.3390/diagnostics10020084 www.mdpi.com/journal/diagnostics. Molecular targeted therapy is most important for adenocarcinoma, and the importance of molecular testing for the purpose of drug adaptation including companion diagnosis has been emphasized. The molecular diagnosis and treatment of NSCLC have evolved due to the development of molecular analysis for targeted therapy including epidermal growth factor receptor (EGFR) mutations [1], anaplastic lymphoma kinase (ALK) gene rearrangements [2], and ROS proto-oncogene 1 receptor tyrosine kinase (ROS1) rearrangements [3]. Formaldehyde-added fixation for LBC presents a difficult challenge for molecular-based testing, because of extensive crosslinking between cellular proteins and/or nucleic acids and nucleic acid degradation which could disrupt the polymerase chain reaction DNA amplification
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