Targeted Next-Generation Sequencing from Fine Needle Aspirates
Hope E Karnes, Eric J Duncavage, Cory T Bernadt. Washington University in St Louis, St. Louis, MO
Background: Molecular testing of cancer is increasingly critical to medical care. Next-generation sequencing (NGS) provides comprehensive, unbiased, and inexpensive mutation analysis of multiple target genes with a single test. However, the utility of NGS on fine needle aspirate (FNA) material, which is often the only specimen available, is unknown. Non-small cell lung cancer (NSCLC) is an ideal model in which to evaluate the application of NGS to cytopathology since FNA is frequently used for diagnosis and staging, and specific molecular therapeutic targets have been identified in NSCLC. We evaluated the performance and quality of targeted NGS in FNAs from a small series of lung adenocarcinomas.
Design: Sequence data were generated from FNAs (percutaneous CT-guided or endobronchial) and paired formalin-fixed paraffin-embedded (FFPE) tissue resections from 5 patients with lung adenocarcinoma. DNA was isolated from cells scraped from both Diff-Quik (DQ) and Papanicolaou (Pap) stained FNA slides. Indexed sequencing libraries were prepared from specimens with ≥ 100 ng DNA. Multiplex, paired-end sequencing of 27 cancer-related genes was performed after hybrid capture enrichment. A custom pipeline based on the Genome Analysis Toolkit was used to identify unique genomic variants from mapped reads. Read quality metrics and single nucleotide variant (SNV) calls were compared across sample types.
Results: Average concordance of single nucleotide variants across specimen types was >99.99%. A small difference (p=0.028) in the percentage of total mapped reads between DQ (98.45%) and FFPE (99.03%) specimens was observed; however, the percentages of on target and duplicate reads did not differ statistically (p> 0.05) between FFPE and cytologic preparations. Between the DQ and Pap preparations there were no differences in mapped reads, on target reads, duplicate reads, or sequence variant calls.
Conclusions: DNA derived from routine FNA fixatives performs comparably to FFPE derived tissue in NGS assays. DNA isolated from the two main types of FNA specimens, DQ and Pap stained slides, yields comprehensive and accurate sequence information, which is statistically indistinguishable from that obtained from FFPE tissue. These results demonstrate the utility of FNAs to provide extensive, high-quality molecular characterization of tumors and support the integration of NGS technologies into the standard cytopathology workflow.
Monday, March 4, 2013 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 54, Monday Morning