Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies

Kevin E. Fisher, Linsheng Zhang, Jason Wang, Geoffrey H. Smith, Scott Newman, Thomas M. Schneider, Rathi N. Pillai, Ragini R. Kudchadkar, Taofeek K. Owonikoko, Suresh S. Ramalingam, David H. Lawson, Keith A. Delman, Bassel F. El-Rayes, Malania M. Wilson, H. Clifford Sullivan, Annie S. Morrison, Serdar Balci, N. Volkan Adsay, Anthony A. Gal, Gabriel L. SicaDebra F. Saxe, Karen P. Mann, Charles E. Hill, Fadlo R. Khuri, Michael R. Rossi

Research output: Contribution to journalArticle

33 Scopus citations

Abstract

We tested and clinically validated a targeted next-generation sequencing (NGS) mutation panel using 80 formalin-fixed, paraffin-embedded (FFPE) tumor samples. Forty non-small cell lung carcinoma (NSCLC), 30 melanoma, and 30 gastrointestinal (12 colonic, 10 gastric, and 8 pancreatic adenocarcinoma) FFPE samples were selected from laboratory archives. After appropriate specimen and nucleic acid quality control, 80 NGS libraries were prepared using the Illumina TruSight tumor (TST) kit and sequenced on the Illumina MiSeq. Sequence alignment, variant calling, and sequencing quality control were performed using vendor software and laboratory-developed analysis workflows. TST generated ≥500× coverage for 98.4% of the 13,952 targeted bases. Reproducible and accurate variant calling was achieved at ≥5% variant allele frequency with 8 to 12 multiplexed samples per MiSeq flow cell. TST detected 112 variants overall, and confirmed all known single-nucleotide variants (n = 27), deletions (n = 5), insertions (n = 3), and multinucleotide variants (n = 3). TST detected at least one variant in 85.0% (68/80), and two or more variants in 36.2% (29/80), of samples. TP53 was the most frequently mutated gene in NSCLC (13 variants; 13/32 samples), gastrointestinal malignancies (15 variants; 13/25 samples), and overall (30 variants; 28/80 samples). BRAF mutations were most common in melanoma (nine variants; 9/23 samples). Clinically relevant NGS data can be obtained from routine clinical FFPE solid tumor specimens using TST, benchtop instruments, and vendor-supplied bioinformatics pipelines.

Original languageEnglish (US)
Pages (from-to)299-315
Number of pages17
JournalJournal of Molecular Diagnostics
Volume18
Issue number2
DOIs
StatePublished - Mar 1 2016

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Medicine

Fingerprint Dive into the research topics of 'Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies'. Together they form a unique fingerprint.

  • Cite this

    Fisher, K. E., Zhang, L., Wang, J., Smith, G. H., Newman, S., Schneider, T. M., Pillai, R. N., Kudchadkar, R. R., Owonikoko, T. K., Ramalingam, S. S., Lawson, D. H., Delman, K. A., El-Rayes, B. F., Wilson, M. M., Sullivan, H. C., Morrison, A. S., Balci, S., Adsay, N. V., Gal, A. A., ... Rossi, M. R. (2016). Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies. Journal of Molecular Diagnostics, 18(2), 299-315. https://doi.org/10.1016/j.jmoldx.2015.11.006