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

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.