Validation of a Next-Generation Sequencing Assay for Clinical Molecular Oncology

被引：152

作者：

Cottrell, Catherine E. ^{[1
,2
]}

Al-Kateb, Hussam ^{[1
,2
]}

Bredemeyer, Andrew J. ^{[1
,2
]}

Duncavage, Eric J. ^{[1
,2
]}

Spencer, David H. ^{[1
,2
]}

Abel, Haley J. ^{[3
,4
]}

Lockwood, Christina M. ^{[1
,2
]}

Hagemann, Ian S. ^{[1
,2
]}

O'Guin, Stephanie M. ^{[3
,4
]}

Burcea, Lauren C. ^{[3
,4
]}

Sawyer, Christopher S. ^{[3
,4
]}

Oschwald, Dayna M. ^{[3
,4
]}

Stratman, Jennifer L. ^{[1
,2
]}

Sher, Done A. ^{[1
,2
]}

Johnson, Mark R. ^{[3
,4
]}

Brown, Justin T. ^{[3
,4
]}

Cliften, Paul F. ^{[3
,4
]}

George, Bijoy ^{[1
,2
]}

McIntosh, Leslie D. ^{[1
,2
]}

Shrivastava, Savita ^{[1
,2
]}

Nguyen, TuDung T. ^{[1
,2
]}

Payton, Jacqueline E. ^{[1
,2
]}

Watson, Mark A. ^{[1
,2
]}

Crosby, Seth D. ^{[3
]}

Head, Richard D. ^{[3
,4
]}

Mitra, Robi D. ^{[3
,4
]}

Nagarajan, Rakesh ^{[1
,2
]}

Kulkarni, Shashikant ^{[1
,2
,3
,4
]}

Seibert, Karen ^{[1
,2
]}

Virgin, Herbert W. ^{[1
,2
]}

Milbrandt, Jeffrey ^{[3
,4
]}

Pfeifer, John D. ^{[1
,2
]}

机构：

[1] Washington Univ, Sch Med, Dept Pathol, St Louis, MO 63110 USA

[2] Washington Univ, Sch Med, Dept Immunol, St Louis, MO 63110 USA

[3] Washington Univ, Sch Med, Dept Genet, St Louis, MO 63110 USA

[4] Washington Univ, Sch Med, Genom & Pathol Serv, St Louis, MO 63110 USA

来源：

JOURNAL OF MOLECULAR DIAGNOSTICS | 2014年 / 16卷 / 01期

关键词：

CELL LUNG-CANCER; MUTATIONS; GENOME; PERFORMANCE; RESISTANCE; GEFITINIB; FRAMEWORK; TUMORS; GENE; BIAS;

D O I：

10.1016/j.jmoldx.2013.10.002

中图分类号：

R36 [病理学];

学科分类号：

100104 ;

摘要：

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 (>= 1000x 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.

引用

页码：89 / 105

页数：17

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