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

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. Cytology smears and liquid‐based preparations provide adequate DNA for next‐generation sequencing at a rate that is comparable to the rate of core biopsies or cell blocks. Significant differences in quality metrics suggest that cytology smears and liquid‐based preparations provide higher quality DNA than formalin‐fixed material.