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

Date Published: June 14, 2019

Publisher: Public Library of Science

Author(s): Toshiaki Akahane, Tomomi Yamaguchi, Yasutaka Kato, Seiya Yokoyama, Taiji Hamada, Yukari Nishida, Michiyo Higashi, Hiroshi Nishihara, Shinsuke Suzuki, Shinichi Ueno, Akihide Tanimoto, Giancarlo Troncone.


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 genome-based cancer medicine.

Partial Text

Tissue samples taken for pathological diagnosis, including fresh, frozen and formalin-fixed tissues, are often used for comprehensive cancer genome analysis. Recent technical developments, especially high-throughput sequencing, enables researchers to subject formalin-fixed paraffin-embedded (FFPE) tissues to cancer genome analysis using next generation sequencing (NGS) [1–4]. A major advantage of FFPE tissues over frozen tissue is that pathologists can observe the cancer lesions in the same region of the samples that is subjected to genome analysis to directly and accurately compare the histological findings and genomic profiles [5]. Using the appropriate tissue fixation with 10% phosphate-buffered neutral formalin, the DNA fragmentation would be minimal even after 5-year storage as paraffin blocks, and whole exome sequence has been successfully performed [6]. The utilization of FFPE tissues for genome sequencing might be less expensive than that of fresh frozen tissues, which requires a special facility such as deep freezers and liquid nitrogen tanks for the long-term storage of samples.

In the present study, we clearly demonstrated that DNA obtained from cultured cells and clinical LBC specimens fixed with CytoRich Red solution were suitable for cancer genome analysis using NGS, even after the samples were stored for up to 1 year. The DNA quality of DNA extracted using four different DNA extraction methods was comparable, and all the samples were suitable for the construction of NGS libraries and for cancer genome analysis.