Targeted next-generation sequencing using fine-needle aspirates from adenocarcinomas of the lung.
BACKGROUND
Molecular testing of cancer is increasingly critical to medicine. Next-generation sequencing (NGS) provides comprehensive, unbiased, and inexpensive mutation analysis of multiple genes with a single test. However, to the authors' knowledge, the usefulness of NGS in fine-needle aspiration (FNA) specimens, which may be the only specimens available, is unknown. Non-small cell lung cancer (NSCLC) is an ideal model in which to evaluate cytopathologic applications of NGS because FNA is used for diagnosis and staging and specific molecular therapeutic targets in NSCLC are known. Herein, the performance and quality of targeted NGS in FNA specimens from a small series of lung adenocarcinomas is evaluated.


METHODS
Sequence data were generated from FNA specimens and paired formalin-fixed paraffin-embedded (FFPE) tissues from 5 patients with lung adenocarcinoma. DNA was isolated from FNA aspirate smears and cores of FFPE tissue. Multiplex sequencing of 27 cancer-related genes was performed after hybrid capture enrichment. Read-quality metrics and single-nucleotide variant calls were compared.


RESULTS
The overall concordance of total reads across specimens was > 99% and the average concordance of single-nucleotide variants was 99.5%. The total reads generated, as well as the percentages of mapped, on-target, and unique reads were statistically indistinguishable (P > 0.05) between FFPE and FNA preparations. There also was no difference in the depth of sequencing coverage, including exon-level coverage in known lung cancer mutation hotspots.


CONCLUSIONS
DNA isolated from FNA slides yields comprehensive, accurate, and statistically indistinguishable sequence information compared with that obtained from FFPE tissue. These results support the integration of NGS technologies into the standard cytopathology workflow. Cancer (Cancer Cytopathol) 2014;122:104-13. © 2013 American Cancer Society.
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri.
Hope E
HE
Karnes
Eric J
EJ
Duncavage
Cory T
CT
Bernadt
D000230:Adenocarcinoma / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D000077192:Adenocarcinoma of Lung; D044963:Biopsy, Fine-Needle / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D017422:Sequence Analysis, DNA / Q000379:methods*
KRAS; epidermal growth factor receptor (EGFR); fine-needle aspiration; lung adenocarcinoma; molecular testing; next-generation sequencing