Molecular Analysis of Liquid-Based Cytological Specimen Using Virtually Positive Sputum with Adenocarcinoma Cells

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.

Next‐generation sequencing in residual liquid‐based cytology specimens for cancer genome analysis

“…A major contributing factor, however, is the amount of tumor fraction in the total sample collected from patient. The use of density reagent to separate and removes non‐tumor cells by density‐gradient centrifugation, 17 did not improve results. Thus, a more specific and effective method to collect tumor cells and exclude non‐tumor cells would improve the availability of LBC samples for molecular testing.…”

Evaluation of DNA and RNA quality from archival formalin‐fixed paraffin‐embedded tissue for next‐generation sequencing – Retrospective study in Japanese single institution

“…However, the Ct value and detection sensitivity of target genes for DNA and RNA were improved by devising means for extracting DNA and RNA. 28,29 The effect of formalin on the PCR method is not only the fragmentation of DNA and RNA, but also the cross-linking formation by formaldehyde. 30 The FFPE tissue samples are an important material used to archive the molecular analyses of malignant tumors.…”

Evaluation of DNA/RNA quality from cell block of malignant mesothelioma and lung adenocarcinoma

“…In formalin‐loaded fixative solutions, extensive crosslinking of cellular proteins and nucleic acids may occur, remarkably decreasing nucleic acid detection sensitivity 23 . Therefore, the use of a nucleic acid extraction kit for FFPE for nucleic acid extraction from LBC specimens derived from sputum greatly improved sensitivity and facilitated the detection of EGFR mutations as a companion diagnosis of lung carcinoma and NGS analysis 24 …”