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

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 × 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.

Next‐generation sequencing in residual liquid‐based cytology specimens for cancer genome analysis

“…9 Our previous study demonstrated that LBC specimens can preserve the DNA quality for NSG analysis even after 5 years of storage as compared to FFPE tissues. 10 Therefore, residual LBC specimens would potentially serve as an alternative source for molecular testing in the diagnosis of cancers. However, in comparison with smears and CBs, residual LBC specimens have some disadvantages for NGS appilication.…”

Next-Generation Sequencing Analysis of Endometrial Screening Liquid-Based Cytology Specimens A Comparative Study to Tissue Specimens

“…To date, LBC represents a routine endometrial cytology method for cancer diagnosis and gene analysis [22]. We also previously reported that residual LBC samples could better preserve the genome quality for downstream NSG, as compared to formalin-xed para n-embedded (FFPE) tissues, even after several years of storage [23]. In the present study, we established a small custom cancer gene panel with full exon coverage of 60 cancer-related genes and 17 microsatellite foci.…”

“…Very similar ndings were obtained from cases no. 20 and 22, and both the TMB and MSI scores were over the cut-off values in these cases.Correlation of the genetic diagnosis from LBC and FFPE specimensIn 10 cases (case nos 9,12,14,16,18,19,[21][22][23][24],. the FFPE tissues from biopsy and/or resection along with endometrial LBC, were subjected to gene panel analysis.…”

Targeted next-generation sequencing assays using triplet samples of normal breast tissue, primary breast cancer, and recurrent/metastatic lesions

“…id=aae35658-5ef2-44b2-bd02-fbe73fe7737c&lang=en). DNA ampli cation for library construction was performed by quantitative polymerase chain reaction (qPCR) using QIAseq DNA QuantiMIZE Assay Kits (#DNQC-100Y-R; Qiagen, Hilden, Germany) [15].…”