Deep targeted sequencing of cytological tumor cells using whole genome amplification.

Abstract

Genomic profiling in lung cancer is essential for precision medicine. Cytological specimens provide an alternative to formalin-fixed paraffin-embedded (FFPE) samples for comprehensive genomic analysis. However, this approach remains challenging when a limited number of tumor cells are available. We applied whole genome amplification (WGA) to cytology specimens to overcome this limitation. Using a lung cancer panel targeting 58 genes, we performed next-generation sequencing of whole genome-amplified DNA extracted from cytological specimens containing 10-20 tumor cells (cyto-WGA) and DNA from corresponding FFPE tumor tissue. We compared sequencing data from cyto-WGA and FFPE samples to examine the detection accuracy of copy number variations and oncogenic and drug-matched variants. The DNA quality and quantity from cyto-WGA were higher than those from FFPE samples (p<.0005 and p<.05, respectively). Sequencing metrics of cyto-WGA and FFPE tissues showed no difference in the number of mapped reads and mean coverage depth, but there were significant differences in the on-target rate (p<.05) and uniformity (p<.0005). Copy number variations in cyto-WGA samples (n=211) were higher than in FFPE samples (n=9) (p<.0001). Thirty-seven oncogenic variants were detected in cyto-WGA and 48 in FFPE. Of these variants, 33 (87%) were present in both samples. In addition, all 16 drug-matched variants were detected in FFPE and cyto-WGA samples with 100% concordance. Cyto-WGA can be a feasible and alternative method to detect oncogenic and drug-matched variants.