TY - JOUR
T1 - Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies
AU - Fisher, Kevin E.
AU - Zhang, Linsheng
AU - Wang, Jason
AU - Smith, Geoffrey H.
AU - Newman, Scott
AU - Schneider, Thomas M.
AU - Pillai, Rathi N.
AU - Kudchadkar, Ragini R.
AU - Owonikoko, Taofeek K.
AU - Ramalingam, Suresh S.
AU - Lawson, David H.
AU - Delman, Keith A.
AU - El-Rayes, Bassel F.
AU - Wilson, Malania M.
AU - Sullivan, H. Clifford
AU - Morrison, Annie S.
AU - Balci, Serdar
AU - Adsay, N. Volkan
AU - Gal, Anthony A.
AU - Sica, Gabriel L.
AU - Saxe, Debra F.
AU - Mann, Karen P.
AU - Hill, Charles E.
AU - Khuri, Fadlo R.
AU - Rossi, Michael R.
N1 - Funding Information:
We thank Jordan Magee, Abiodun Ojo, and Heather Jones for their excellent technical assistance.
Funding Information:
Supported by institutional funding and in part by the Emory Integrated Genomics Core and Biostatistics and Bioinformatics Shared resource of Winship Cancer Institute of Emory University and NIH/National Cancer Institute award P30CA138292.
Publisher Copyright:
© 2016 American Society for Investigative Pathology and the Association for Molecular Pathology.
PY - 2016/3/1
Y1 - 2016/3/1
N2 - 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.
AB - 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.
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U2 - 10.1016/j.jmoldx.2015.11.006
DO - 10.1016/j.jmoldx.2015.11.006
M3 - Article
C2 - 26801070
AN - SCOPUS:84960939822
VL - 18
SP - 299
EP - 315
JO - Journal of Molecular Diagnostics
JF - Journal of Molecular Diagnostics
SN - 1525-1578
IS - 2
ER -