Nishikawa Takeshi, Fujii Tomomi, Tatsumi Shigenobu, Sugimoto Aya, Sekita-Hatakeyama Yoko, Shimada Keiji, Yamazaki Masaharu, Hatakeyama Kinta, Ohbayashi Chiho
Department of Diagnostic Pathology, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8521, Japan.
Department of Diagnostic Pathology, Nara City Hospital, Nara 630-8305, Japan.
Diagnostics (Basel). 2020 Feb 5;10(2):84. doi: 10.3390/diagnostics10020084.
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.
痰液的液基细胞学（LBC）分析是检测肺癌的一种有用的诊断和预后工具。源自肺癌细胞的DNA和RNA可用于该诊断。然而，由于市售固定试剂盒中福尔马林的影响，细胞学材料的质量并不总是足以进行分子分析。在本研究中，我们使用肺癌细胞系和痰液，研究了用于福尔马林固定的LBC分析的DNA和RNA提取方法。人非小细胞肺癌细胞系用LBC固定试剂（如CytoRich红色防腐剂）固定。通过定量PCR（qPCR）和基于荧光共振能量转移（FRET）的优先同源双链体形成分析（F-PHFA）方法，分别对HCC827、H1975和H1299细胞中的甲状腺转录因子-1（TTF-1）和肌动蛋白mRNA、表皮生长因子受体（EGFR）DNA进行定量，并对HCC827和H1975细胞中的EGFR进行突变分析。使用用于福尔马林固定石蜡包埋（FFPE）的RNA和/或DNA提取试剂盒，从用各种LBC溶液固定的细胞系中提取的mRNA和DNA，通过qPCR可有效检测。LBC中EGFR突变的检测限为5%的突变阳性细胞率。在痰液中，HCC827中EGFR外显子19缺失的检测限在超过1.5%的阳性细胞中被检测到。相比之下，H1975中T790M/L858R突变的检测限在超过13%的阳性细胞中被检测到。我们还使用下一代测序（NGS）检测EGFR突变。NGS对EGFR突变的检测限低于F-PHFA方法。此外，超过0.1%的阳性细胞可以通过细胞形态学检测到。我们的结果表明，LBC系统是细胞病理学和基因分析的强大工具。然而，在LBC检测的EGFR突变的基因分析中，应仔细注意假阴性结果的发生率。
