TY - JOUR
T1 - Validation of a next-generation sequencing assay for clinical molecular oncology
AU - Cottrell, Catherine E.
AU - Al-Kateb, Hussam
AU - Bredemeyer, Andrew J.
AU - Duncavage, Eric J.
AU - Spencer, David H.
AU - Abel, Haley J.
AU - Lockwood, Christina M.
AU - Hagemann, Ian S.
AU - O'Guin, Stephanie M.
AU - Burcea, Lauren C.
AU - Sawyer, Christopher S.
AU - Oschwald, Dayna M.
AU - Stratman, Jennifer L.
AU - Sher, Dorie A.
AU - Johnson, Mark R.
AU - Brown, Justin T.
AU - Cliften, Paul F.
AU - George, Bijoy
AU - McIntosh, Leslie D.
AU - Shrivastava, Savita
AU - Nguyen, Tudung T.
AU - Payton, Jacqueline E.
AU - Watson, Mark A.
AU - Crosby, Seth D.
AU - Head, Richard D.
AU - Mitra, Robi D.
AU - Nagarajan, Rakesh
AU - Kulkarni, Shashikant
AU - Seibert, Karen
AU - Virgin IV, Herbert W.
AU - Milbrandt, Jeffrey
AU - Pfeifer, John D.
N1 - Funding Information:
Supported by Genomics and Pathology Services, Washington University School of Medicine, St. Louis, MO.
PY - 2014/1
Y1 - 2014/1
N2 - Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management.
AB - Currently, oncology testing includes molecular studies and cytogenetic analysis to detect genetic aberrations of clinical significance. Next-generation sequencing (NGS) allows rapid analysis of multiple genes for clinically actionable somatic variants. The WUCaMP assay uses targeted capture for NGS analysis of 25 cancer-associated genes to detect mutations at actionable loci. We present clinical validation of the assay and a detailed framework for design and validation of similar clinical assays. Deep sequencing of 78 tumor specimens (≥1000× average unique coverage across the capture region) achieved high sensitivity for detecting somatic variants at low allele fraction (AF). Validation revealed sensitivities and specificities of 100% for detection of single-nucleotide variants (SNVs) within coding regions, compared with SNP array sequence data (95% CI = 83.4-100.0 for sensitivity and 94.2-100.0 for specificity) or whole-genome sequencing (95% CI = 89.1-100.0 for sensitivity and 99.9-100.0 for specificity) of HapMap samples. Sensitivity for detecting variants at an observed 10% AF was 100% (95% CI = 93.2-100.0) in HapMap mixes. Analysis of 15 masked specimens harboring clinically reported variants yielded concordant calls for 13/13 variants at AF of ≥15%. The WUCaMP assay is a robust and sensitive method to detect somatic variants of clinical significance in molecular oncology laboratories, with reduced time and cost of genetic analysis allowing for strategic patient management.
UR - http://www.scopus.com/inward/record.url?scp=84890421610&partnerID=8YFLogxK
U2 - 10.1016/j.jmoldx.2013.10.002
DO - 10.1016/j.jmoldx.2013.10.002
M3 - Article
C2 - 24211365
AN - SCOPUS:84890421610
SN - 1525-1578
VL - 16
SP - 89
EP - 105
JO - Journal of Molecular Diagnostics
JF - Journal of Molecular Diagnostics
IS - 1
ER -