Development and validation of an ultradeep next-generation sequencing assay for testing of plasma cell-free DNA from patients with advanced cancer

Filip Janku, Shile Zhang, Jill Waters, Li Liu, Helen J. Huang, Vivek Subbiah, David S. Hong, Daniel D. Karp, Siqing Fu, Xuyu Cai, Nishma M. Ramzanali, Kiran Madwani, Goran Cabrilo, Debra L. Andrews, Yue Zhao, Milind Javle, E. Scott Kopetz, Rajyalakshmi Luthra, Hyunsung J. Kim, Sante GnerreRavi Vijaya Satya, Han Yu Chuang, Kristina M. Kruglyak, Jonathan Toung, Chen Zhao, Richard Shen, John V. Heymach, Funda Meric-Bernstam, Gordon Mills, Jian Bing Fan, Neeraj S. Salathia

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Purpose: Tumor-derived cell-free DNA (cfDNA) in plasma can be used for molecular testing and provide an attractive alternative to tumor tissue. Commonly used PCR-based technologies can test for limited number of alterations at the time. Therefore, novel ultrasensitive technologies capable of testing for a broad spectrum of molecular alterations are needed to further personalized cancer therapy. Experimental Design: We developed a highly sensitive ultradeep next-generation sequencing (NGS) assay using reagents from TruSeqNano library preparation and NexteraRapid Capture target enrichment kits to generate plasma cfDNA sequencing libraries for mutational analysis in 61 cancer-related genes using common bioinformatics tools. The results were retrospectively compared with molecular testing of archival primary or metastatic tumor tissue obtained at different points of clinical care. Results: In a study of 55 patients with advanced cancer, the ultradeep NGS assay detected 82% (complete detection) to 87% (complete and partial detection) of the aberrations identified in discordantly collected corresponding archival tumor tissue. Patients with a low variant allele frequency (VAF) of mutant cfDNA survived longer than those with a high VAF did (P = 0.018). In patients undergoing systemic therapy, radiological response was positively associated with changes in cfDNA VAF (P = 0.02), and compared with unchanged/increased mutant cfDNA VAF, decreased cfDNA VAF was associated with longer time to treatment failure (TTF; P = 0.03). Conclusions: Ultradeep NGS assay has good sensitivity compared with conventional clinical mutation testing of archival specimens. A high VAF in mutant cfDNA corresponded with shorter survival. Changes in VAF of mutated cfDNA were associated with TTF.

Original languageEnglish (US)
Pages (from-to)5648-5656
Number of pages9
JournalClinical Cancer Research
Volume23
Issue number18
DOIs
StatePublished - Sep 15 2017
Externally publishedYes

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Plasma Cells
Gene Frequency
DNA
Neoplasms
Point-of-Care Systems
Technology
Neoplasm Genes
Computational Biology
Treatment Failure
Gene Library
DNA Sequence Analysis
Libraries
Research Design
Polymerase Chain Reaction
Mutation
Survival
Therapeutics

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Development and validation of an ultradeep next-generation sequencing assay for testing of plasma cell-free DNA from patients with advanced cancer. / Janku, Filip; Zhang, Shile; Waters, Jill; Liu, Li; Huang, Helen J.; Subbiah, Vivek; Hong, David S.; Karp, Daniel D.; Fu, Siqing; Cai, Xuyu; Ramzanali, Nishma M.; Madwani, Kiran; Cabrilo, Goran; Andrews, Debra L.; Zhao, Yue; Javle, Milind; Kopetz, E. Scott; Luthra, Rajyalakshmi; Kim, Hyunsung J.; Gnerre, Sante; Satya, Ravi Vijaya; Chuang, Han Yu; Kruglyak, Kristina M.; Toung, Jonathan; Zhao, Chen; Shen, Richard; Heymach, John V.; Meric-Bernstam, Funda; Mills, Gordon; Fan, Jian Bing; Salathia, Neeraj S.

In: Clinical Cancer Research, Vol. 23, No. 18, 15.09.2017, p. 5648-5656.

Research output: Contribution to journalArticle

Janku, F, Zhang, S, Waters, J, Liu, L, Huang, HJ, Subbiah, V, Hong, DS, Karp, DD, Fu, S, Cai, X, Ramzanali, NM, Madwani, K, Cabrilo, G, Andrews, DL, Zhao, Y, Javle, M, Kopetz, ES, Luthra, R, Kim, HJ, Gnerre, S, Satya, RV, Chuang, HY, Kruglyak, KM, Toung, J, Zhao, C, Shen, R, Heymach, JV, Meric-Bernstam, F, Mills, G, Fan, JB & Salathia, NS 2017, 'Development and validation of an ultradeep next-generation sequencing assay for testing of plasma cell-free DNA from patients with advanced cancer', Clinical Cancer Research, vol. 23, no. 18, pp. 5648-5656. https://doi.org/10.1158/1078-0432.CCR-17-0291
Janku, Filip ; Zhang, Shile ; Waters, Jill ; Liu, Li ; Huang, Helen J. ; Subbiah, Vivek ; Hong, David S. ; Karp, Daniel D. ; Fu, Siqing ; Cai, Xuyu ; Ramzanali, Nishma M. ; Madwani, Kiran ; Cabrilo, Goran ; Andrews, Debra L. ; Zhao, Yue ; Javle, Milind ; Kopetz, E. Scott ; Luthra, Rajyalakshmi ; Kim, Hyunsung J. ; Gnerre, Sante ; Satya, Ravi Vijaya ; Chuang, Han Yu ; Kruglyak, Kristina M. ; Toung, Jonathan ; Zhao, Chen ; Shen, Richard ; Heymach, John V. ; Meric-Bernstam, Funda ; Mills, Gordon ; Fan, Jian Bing ; Salathia, Neeraj S. / Development and validation of an ultradeep next-generation sequencing assay for testing of plasma cell-free DNA from patients with advanced cancer. In: Clinical Cancer Research. 2017 ; Vol. 23, No. 18. pp. 5648-5656.
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T1 - Development and validation of an ultradeep next-generation sequencing assay for testing of plasma cell-free DNA from patients with advanced cancer

AU - Janku, Filip

AU - Zhang, Shile

AU - Waters, Jill

AU - Liu, Li

AU - Huang, Helen J.

AU - Subbiah, Vivek

AU - Hong, David S.

AU - Karp, Daniel D.

AU - Fu, Siqing

AU - Cai, Xuyu

AU - Ramzanali, Nishma M.

AU - Madwani, Kiran

AU - Cabrilo, Goran

AU - Andrews, Debra L.

AU - Zhao, Yue

AU - Javle, Milind

AU - Kopetz, E. Scott

AU - Luthra, Rajyalakshmi

AU - Kim, Hyunsung J.

AU - Gnerre, Sante

AU - Satya, Ravi Vijaya

AU - Chuang, Han Yu

AU - Kruglyak, Kristina M.

AU - Toung, Jonathan

AU - Zhao, Chen

AU - Shen, Richard

AU - Heymach, John V.

AU - Meric-Bernstam, Funda

AU - Mills, Gordon

AU - Fan, Jian Bing

AU - Salathia, Neeraj S.

PY - 2017/9/15

Y1 - 2017/9/15

N2 - Purpose: Tumor-derived cell-free DNA (cfDNA) in plasma can be used for molecular testing and provide an attractive alternative to tumor tissue. Commonly used PCR-based technologies can test for limited number of alterations at the time. Therefore, novel ultrasensitive technologies capable of testing for a broad spectrum of molecular alterations are needed to further personalized cancer therapy. Experimental Design: We developed a highly sensitive ultradeep next-generation sequencing (NGS) assay using reagents from TruSeqNano library preparation and NexteraRapid Capture target enrichment kits to generate plasma cfDNA sequencing libraries for mutational analysis in 61 cancer-related genes using common bioinformatics tools. The results were retrospectively compared with molecular testing of archival primary or metastatic tumor tissue obtained at different points of clinical care. Results: In a study of 55 patients with advanced cancer, the ultradeep NGS assay detected 82% (complete detection) to 87% (complete and partial detection) of the aberrations identified in discordantly collected corresponding archival tumor tissue. Patients with a low variant allele frequency (VAF) of mutant cfDNA survived longer than those with a high VAF did (P = 0.018). In patients undergoing systemic therapy, radiological response was positively associated with changes in cfDNA VAF (P = 0.02), and compared with unchanged/increased mutant cfDNA VAF, decreased cfDNA VAF was associated with longer time to treatment failure (TTF; P = 0.03). Conclusions: Ultradeep NGS assay has good sensitivity compared with conventional clinical mutation testing of archival specimens. A high VAF in mutant cfDNA corresponded with shorter survival. Changes in VAF of mutated cfDNA were associated with TTF.

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