Cargando…

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

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Akahane, Toshiaki, Yamaguchi, Tomomi, Kato, Yasutaka, Yokoyama, Seiya, Hamada, Taiji, Nishida, Yukari, Higashi, Michiyo, Nishihara, Hiroshi, Suzuki, Shinsuke, Ueno, Shinichi, Tanimoto, Akihide
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6568385/
https://www.ncbi.nlm.nih.gov/pubmed/31199826
http://dx.doi.org/10.1371/journal.pone.0217724
_version_ 1783427237302763520
author Akahane, Toshiaki
Yamaguchi, Tomomi
Kato, Yasutaka
Yokoyama, Seiya
Hamada, Taiji
Nishida, Yukari
Higashi, Michiyo
Nishihara, Hiroshi
Suzuki, Shinsuke
Ueno, Shinichi
Tanimoto, Akihide
author_facet Akahane, Toshiaki
Yamaguchi, Tomomi
Kato, Yasutaka
Yokoyama, Seiya
Hamada, Taiji
Nishida, Yukari
Higashi, Michiyo
Nishihara, Hiroshi
Suzuki, Shinsuke
Ueno, Shinichi
Tanimoto, Akihide
author_sort Akahane, Toshiaki
collection PubMed
description 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 × 10(3) or 2 × 10(4) 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 × 10(3) 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.
format Online
Article
Text
id pubmed-6568385
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-65683852019-06-20 Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis Akahane, Toshiaki Yamaguchi, Tomomi Kato, Yasutaka Yokoyama, Seiya Hamada, Taiji Nishida, Yukari Higashi, Michiyo Nishihara, Hiroshi Suzuki, Shinsuke Ueno, Shinichi Tanimoto, Akihide PLoS One Research Article 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 × 10(3) or 2 × 10(4) 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 × 10(3) 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. Public Library of Science 2019-06-14 /pmc/articles/PMC6568385/ /pubmed/31199826 http://dx.doi.org/10.1371/journal.pone.0217724 Text en © 2019 Akahane et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Akahane, Toshiaki
Yamaguchi, Tomomi
Kato, Yasutaka
Yokoyama, Seiya
Hamada, Taiji
Nishida, Yukari
Higashi, Michiyo
Nishihara, Hiroshi
Suzuki, Shinsuke
Ueno, Shinichi
Tanimoto, Akihide
Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis
title Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis
title_full Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis
title_fullStr Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis
title_full_unstemmed Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis
title_short Comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis
title_sort comprehensive validation of liquid-based cytology specimens for next-generation sequencing in cancer genome analysis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6568385/
https://www.ncbi.nlm.nih.gov/pubmed/31199826
http://dx.doi.org/10.1371/journal.pone.0217724
work_keys_str_mv AT akahanetoshiaki comprehensivevalidationofliquidbasedcytologyspecimensfornextgenerationsequencingincancergenomeanalysis
AT yamaguchitomomi comprehensivevalidationofliquidbasedcytologyspecimensfornextgenerationsequencingincancergenomeanalysis
AT katoyasutaka comprehensivevalidationofliquidbasedcytologyspecimensfornextgenerationsequencingincancergenomeanalysis
AT yokoyamaseiya comprehensivevalidationofliquidbasedcytologyspecimensfornextgenerationsequencingincancergenomeanalysis
AT hamadataiji comprehensivevalidationofliquidbasedcytologyspecimensfornextgenerationsequencingincancergenomeanalysis
AT nishidayukari comprehensivevalidationofliquidbasedcytologyspecimensfornextgenerationsequencingincancergenomeanalysis
AT higashimichiyo comprehensivevalidationofliquidbasedcytologyspecimensfornextgenerationsequencingincancergenomeanalysis
AT nishiharahiroshi comprehensivevalidationofliquidbasedcytologyspecimensfornextgenerationsequencingincancergenomeanalysis
AT suzukishinsuke comprehensivevalidationofliquidbasedcytologyspecimensfornextgenerationsequencingincancergenomeanalysis
AT uenoshinichi comprehensivevalidationofliquidbasedcytologyspecimensfornextgenerationsequencingincancergenomeanalysis
AT tanimotoakihide comprehensivevalidationofliquidbasedcytologyspecimensfornextgenerationsequencingincancergenomeanalysis