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Comparative Study
. 2017 Oct;125(10):786-794.
doi: 10.1002/cncy.21897. Epub 2017 Jul 20.

Next-generation sequencing of cytologic preparations: An analysis of quality metrics

David H Hwang  1 , Elizabeth P Garcia  2 , Matthew D Ducar  3 , Edmund S Cibas  1 , Lynette M Sholl  1   2
Affiliations
Free article
Comparative Study

Next-generation sequencing of cytologic preparations: An analysis of quality metrics

David H Hwang et al. Cancer Cytopathol. 2017 Oct.
Free article

Abstract

Background: Next-generation sequencing (NGS) fails for many small biopsies (BXs) because of a low overall DNA concentration or tumor percentage. Cytology smears and liquid-based preparations (LBPs), or smears/LBPs, often contain abundant tumor cells and may provide adequate material for molecular testing when other materials are insufficient. This study examined the performance of smears/LBPs on a clinical NGS assay.

Methods: This study retrospectively reviewed quality metrics from consecutive smear/LBP, core BX, and cell block (CB) cases run on a hybrid-capture NGS assay interrogating 309 cancer-related genes. The following quality metrics were compared: adequacy rate, initial DNA concentration, postshearing fragment size, post-library preparation fragment size, fragment size difference, insert size, total reads, passing-filter reads aligned, percent passing-filter unique reads aligned, mean target coverage, percentage of loci with >100× coverage, percent duplication rate, percent selected bases, and percent usable bases on bait.

Results: Twenty-three of 26 smears/LBPs (88%) were successfully sequenced, whereas 77 of 87 core BXs (89%) and 29 of 30 CBs (97%) were. The mean target coverage, median insert size, and percent usable bases were significantly higher in the smear/LBP category. The postshearing fragment size and the percent duplication were significantly lower for smears/LBPs.

Conclusions: The adequacy rate of cytology smears/LBPs for NGS is comparable to that of core BXs or CBs. Increased values for the mean insert size, mean target coverage, and percent usable bases, along with a lower duplication rate, suggest that smears/LBPs provide higher quality DNA than formalin-fixed material. Cytology smears/LBPs can serve as a valuable source of material for molecular testing. Cancer Cytopathol 2017;125:786-94. © 2017 American Cancer Society.

Keywords: body fluids; cytology; fine-needle aspiration; formaldehyde; molecular diagnostic techniques.

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