Clinical actionability enhanced through deep targeted sequencing of solid tumors

Ken Chen, Funda Meric-Bernstam, Hao Zhao, Qingxiu Zhang, Nader Ezzeddine, Lin Ya Tang, Yuan Qi, Yong Mao, Tenghui Chen, Zechen Chong, Wanding Zhou, Xiaofeng Zheng, Amber Johnson, Kenneth D. Aldape, Mark J. Routbort, Rajyalakshmi Luthra, Scott Kopetz, Michael A. Davies, John De Groot, Stacy MoulderRavi Vinod, Carol J. Farhangfar, Kenna Mills Shaw, John Mendelsohn, Gordon Mills, Agda Karina Eterovic

Research output: Contribution to journalArticle

63 Citations (Scopus)

Abstract

BACKGROUND: Further advances of targeted cancer therapy require comprehensive in-depth profiling of somatic mutations that are present in subpopulations of tumor cells in a clinical tumor sample. However, it is unclear to what extent such intratumor heterogeneity is present and whether it may affect clinical decision-making. To study this question, we established a deep targeted sequencing platform to identify potentially actionable DNA alterations in tumor samples. METHODS: We assayed 515 formalin-fixed paraffin-embedded (FFPE) tumor samples and matched germline DNA (475 patients) from 11 disease sites by capturing and sequencing all the exons in 201 cancer-related genes. Mutations, indels, and copy number data were reported. RESULTS: We obtained a 1000-fold mean sequencing depth and identified 4794 nonsynonymous mutations in the samples analyzed, of which 15.2% were present at <10% allele frequency. Most of these low level mutations occurred at known oncogenic hotspots and are likely functional. Identifying low level mutations improved identification of mutations in actionable genes in 118 (24.84%) patients, among which 47 (9.8%) otherwise would have been unactionable. In addition, acquiring ultrahigh depth also ensured a low false discovery rate (<2.2%) from FFPE samples. CONCLUSIONS: Our results were as accurate as a commercially available CLIA-compliant hotspot panel but allowed the detection of a higher number of mutations in actionable genes. Our study reveals the critical importance of acquiring and utilizing high sequencing depth in profiling clinical tumor samples and presents a very useful platform for implementing routine sequencing in a cancer care institution.

Original languageEnglish (US)
Pages (from-to)544-553
Number of pages10
JournalClinical Chemistry
Volume61
Issue number3
DOIs
StatePublished - Jan 1 2015
Externally publishedYes

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High-Throughput Nucleotide Sequencing
Tumors
Mutation
Genes
Neoplasms
Paraffin
Formaldehyde
Depth profiling
DNA
Exons
Neoplasm Genes
Decision making
Cells
Gene Frequency

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Chen, K., Meric-Bernstam, F., Zhao, H., Zhang, Q., Ezzeddine, N., Tang, L. Y., ... Eterovic, A. K. (2015). Clinical actionability enhanced through deep targeted sequencing of solid tumors. Clinical Chemistry, 61(3), 544-553. https://doi.org/10.1373/clinchem.2014.231100

Clinical actionability enhanced through deep targeted sequencing of solid tumors. / Chen, Ken; Meric-Bernstam, Funda; Zhao, Hao; Zhang, Qingxiu; Ezzeddine, Nader; Tang, Lin Ya; Qi, Yuan; Mao, Yong; Chen, Tenghui; Chong, Zechen; Zhou, Wanding; Zheng, Xiaofeng; Johnson, Amber; Aldape, Kenneth D.; Routbort, Mark J.; Luthra, Rajyalakshmi; Kopetz, Scott; Davies, Michael A.; De Groot, John; Moulder, Stacy; Vinod, Ravi; Farhangfar, Carol J.; Shaw, Kenna Mills; Mendelsohn, John; Mills, Gordon; Eterovic, Agda Karina.

In: Clinical Chemistry, Vol. 61, No. 3, 01.01.2015, p. 544-553.

Research output: Contribution to journalArticle

Chen, K, Meric-Bernstam, F, Zhao, H, Zhang, Q, Ezzeddine, N, Tang, LY, Qi, Y, Mao, Y, Chen, T, Chong, Z, Zhou, W, Zheng, X, Johnson, A, Aldape, KD, Routbort, MJ, Luthra, R, Kopetz, S, Davies, MA, De Groot, J, Moulder, S, Vinod, R, Farhangfar, CJ, Shaw, KM, Mendelsohn, J, Mills, G & Eterovic, AK 2015, 'Clinical actionability enhanced through deep targeted sequencing of solid tumors', Clinical Chemistry, vol. 61, no. 3, pp. 544-553. https://doi.org/10.1373/clinchem.2014.231100
Chen K, Meric-Bernstam F, Zhao H, Zhang Q, Ezzeddine N, Tang LY et al. Clinical actionability enhanced through deep targeted sequencing of solid tumors. Clinical Chemistry. 2015 Jan 1;61(3):544-553. https://doi.org/10.1373/clinchem.2014.231100
Chen, Ken ; Meric-Bernstam, Funda ; Zhao, Hao ; Zhang, Qingxiu ; Ezzeddine, Nader ; Tang, Lin Ya ; Qi, Yuan ; Mao, Yong ; Chen, Tenghui ; Chong, Zechen ; Zhou, Wanding ; Zheng, Xiaofeng ; Johnson, Amber ; Aldape, Kenneth D. ; Routbort, Mark J. ; Luthra, Rajyalakshmi ; Kopetz, Scott ; Davies, Michael A. ; De Groot, John ; Moulder, Stacy ; Vinod, Ravi ; Farhangfar, Carol J. ; Shaw, Kenna Mills ; Mendelsohn, John ; Mills, Gordon ; Eterovic, Agda Karina. / Clinical actionability enhanced through deep targeted sequencing of solid tumors. In: Clinical Chemistry. 2015 ; Vol. 61, No. 3. pp. 544-553.
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AU - Chen, Ken

AU - Meric-Bernstam, Funda

AU - Zhao, Hao

AU - Zhang, Qingxiu

AU - Ezzeddine, Nader

AU - Tang, Lin Ya

AU - Qi, Yuan

AU - Mao, Yong

AU - Chen, Tenghui

AU - Chong, Zechen

AU - Zhou, Wanding

AU - Zheng, Xiaofeng

AU - Johnson, Amber

AU - Aldape, Kenneth D.

AU - Routbort, Mark J.

AU - Luthra, Rajyalakshmi

AU - Kopetz, Scott

AU - Davies, Michael A.

AU - De Groot, John

AU - Moulder, Stacy

AU - Vinod, Ravi

AU - Farhangfar, Carol J.

AU - Shaw, Kenna Mills

AU - Mendelsohn, John

AU - Mills, Gordon

AU - Eterovic, Agda Karina

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N2 - BACKGROUND: Further advances of targeted cancer therapy require comprehensive in-depth profiling of somatic mutations that are present in subpopulations of tumor cells in a clinical tumor sample. However, it is unclear to what extent such intratumor heterogeneity is present and whether it may affect clinical decision-making. To study this question, we established a deep targeted sequencing platform to identify potentially actionable DNA alterations in tumor samples. METHODS: We assayed 515 formalin-fixed paraffin-embedded (FFPE) tumor samples and matched germline DNA (475 patients) from 11 disease sites by capturing and sequencing all the exons in 201 cancer-related genes. Mutations, indels, and copy number data were reported. RESULTS: We obtained a 1000-fold mean sequencing depth and identified 4794 nonsynonymous mutations in the samples analyzed, of which 15.2% were present at <10% allele frequency. Most of these low level mutations occurred at known oncogenic hotspots and are likely functional. Identifying low level mutations improved identification of mutations in actionable genes in 118 (24.84%) patients, among which 47 (9.8%) otherwise would have been unactionable. In addition, acquiring ultrahigh depth also ensured a low false discovery rate (<2.2%) from FFPE samples. CONCLUSIONS: Our results were as accurate as a commercially available CLIA-compliant hotspot panel but allowed the detection of a higher number of mutations in actionable genes. Our study reveals the critical importance of acquiring and utilizing high sequencing depth in profiling clinical tumor samples and presents a very useful platform for implementing routine sequencing in a cancer care institution.

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