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

Toshiaki Akahane, Tomomi Yamaguchi, Yasutaka Kato, Seiya Yokoyama, Taiji Hamada, Yukari Nishida, Michiyo Higashi, Hiroshi Nishihara, Shinsuke Suzuki, Shinichi Ueno, Akihide Tanimoto

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Abstract

In addition to conventional cytology, liquid-based cytology (LBC) is also used for immunocytochemistry and gene analysis. However, an appropriate method to obtain high quality DNA for next-generation sequencing (NGS) using LBC specimens remains controversial. We determined the optimal conditions for fixation with an alcohol-based fixative for LBC and DNA extraction using cultured cancer cell lines and clinical specimens. The extracted DNA was processed for NGS after the DNA quality was confirmed based on the DNA concentration and degree of degradation. The optimal conditions for cultured cells to obtain high quality DNA were to fix the cells at a density of 6 × 103 or 2 × 104 cells/mL and to use the magnetic bead-based DNA extraction method. Even after storing the fixed cells for 90 days, DNA extracted using the above and other extraction kits, including membrane-based methods, did not undergo degradation. Furthermore, 5-year-old residual LBC samples demonstrated high DNA quality that was suitable for NGS. Furthermore, a cancer genome panel analysis was successfully performed with DNA extracted from cultured cells fixed at 6 × 103 cells/mL for 90 days, and with DNA from residual LBC samples even after 1 year of storage. Residual LBC samples may be a useful source of DNA for clinical NGS to promote genomebased cancer medicine.

Original languageEnglish
Article numbere0217724
JournalPloS one
Volume14
Issue number6
DOIs
Publication statusPublished - 2019 Jun 1

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ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Akahane, T., Yamaguchi, T., Kato, Y., Yokoyama, S., Hamada, T., Nishida, Y., Higashi, M., Nishihara, H., Suzuki, S., Ueno, S., & Tanimoto, A. (2019). Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis. PloS one, 14(6), [e0217724]. https://doi.org/10.1371/journal.pone.0217724