Reducing DNA damage by formaldehyde in liquid‐based cytology preservation solutions to enable the molecular testing of lung cancer specimens

Abstract

Background Liquid‐based cytology (LBC) is a useful cytopathological method, and LBC lung adenocarcinoma specimens may be used for genetic analysis in the near future. In the current study, the authors determined whether LBC specimens can be used for epidermal growth factor receptor (EGFR) mutation analysis in human lung adenocarcinoma cell lines. Methods Genomic DNA was extracted from 3 lung adenocarcinoma cell lines that were fixed in LBC preservation solution using 2 protocols (one for cultured cells and one for tissues) of a DNA extraction kit. Different fixation times were tested for each protocol: 30 minutes, 1 hour, and 1 to 9 days. As controls, cells also were fixed in 10% formalin or 95% ethanol. The authors investigated the effect of fixation time on DNA fragmentation, polymerase chain reaction (PCR) amplification, and EGFR mutation detection. Results The DNA yield of LBC specimens tended to decrease depending on fixation time. When using the DNA extraction protocol for tissues, PCR amplification was successful after 9 days of fixation, although extracted genomic DNA that was fixed for >1 hour demonstrated fragmentation. Mutation analyses using the Cycleave PCR method were successful after 7 days of fixation. The DNA extraction protocol for tissues was appropriate for lung adenocarcinoma cell lines that were stored for >1 day in a preservative solution. The results of the current study demonstrated that EGFR mutations can be detected on day 7 using lung adenocarcinoma cell lines fixed in CytoRich Red preservative. Conclusions When LBC specimens are used for targeted molecular genetic testing, the appropriate preservative solution and extraction protocol first should be determined.

Testing EGFR with Idylla on Cytological Specimens of Lung Cancer: A Review

“…This means that virtually any subsequent test can be performed on the material if it is stored correctly, from cytological (smears) to histological (cell blocks) preparations, with the whole array of ancillary tests at one’s disposal (immunocytochemistry, immunohistochemistry, flow cytometry, cytogenetics, molecular biology) [ 102 , 103 , 104 , 105 , 106 , 107 ]. One important factor that must not be overlooked in the context of molecular biology is that harvesting fresh (unfixed) material means that the quality of nucleic acids (both DNA and RNA) is at its best because no fixatives have been used and the risk of contamination is vastly reduced [ 108 , 109 , 110 ].…”

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.…”

Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis

“…When LBC specimens were fixed with CytoRich Red and DNA was extracted from the cells on glass slides, Kim et al similarly failed to detect BRAF exon 15 (a 226-bp PCR product) [25]. Another study reported poor or no detection of EGFR exon 19 (a 190-bp PCR product) from DNA extracted from cultured cells fixed in CytoRich Red fixative for more than 1 day [26]. The experimental protocol of this previous study was very similar to that of the present study, except that they used a higher cell density of 1 × 10 6 cells/mL for fixation.…”