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Development and Validation of an Ultradeep Next-Generation Sequencing Assay for Testing of Plasma Cell-Free DNA from Patients with Advanced Cancer.

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. Clin Cancer Res; 23(18); 5648-56. ©2017 AACR.

SUBMITTER: Janku F

PROVIDER: S-EPMC5600825 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Development and Validation of an Ultradeep Next-Generation Sequencing Assay for Testing of Plasma Cell-Free DNA from Patients with Advanced Cancer.

Janku Filip F Zhang Shile S Waters Jill J Liu Li L Huang Helen J HJ Subbiah Vivek V Hong David S DS Karp Daniel D DD Fu Siqing S Cai Xuyu X Ramzanali Nishma M NM Madwani Kiran K Cabrilo Goran G Andrews Debra L DL Zhao Yue Y Javle Milind M Kopetz E Scott ES Luthra Rajyalakshmi R Kim Hyunsung J HJ Gnerre Sante S Satya Ravi Vijaya RV Chuang Han-Yu HY Kruglyak Kristina M KM Toung Jonathan J Zhao Chen C Shen Richard R Heymach John V JV Meric-Bernstam Funda F Mills Gordon B GB Fan Jian-Bing JB Salathia Neeraj S NS

Clinical cancer research : an official journal of the American Association for Cancer Research 20170523 18

<b>Purpose:</b> 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.<b>Experimental Design:</b> We developed a highly sensitive ultradeep next-generation sequencing ...[more]

PMID: 28536309

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Project description:BackgroundCharacterization of circulating tumor DNA (ctDNA) has been integrated into clinical practice. Although labs have standardized validation procedures to develop single locus tests, the efficacy of on-site plasma-based next-generation sequencing (NGS) assays still needs to be proved.Materials and methodsIn this retrospective study, we profiled DNA from matched tissue and plasma samples from 75 patients with cancer. We applied an NGS test that detects clinically relevant alterations in 33 genes and microsatellite instability (MSI) to analyze plasma cell-free DNA (cfDNA).ResultsThe concordance between alterations detected in both tissue and plasma samples was higher in patients with metastatic disease. The NGS test detected 77% of sequence alterations, amplifications, and fusions that were found in metastatic samples compared with 45% of those alterations found in the primary tumor samples (p = .00005). There was 87% agreement on MSI status between the NGS test and tumor tissue results. In three patients, MSI-high ctDNA correlated with response to immunotherapy. In addition, the NGS test revealed an FGFR2 amplification that was not detected in tumor tissue from a patient with metastatic gastric cancer, emphasizing the importance of profiling plasma samples in patients with advanced cancer.ConclusionOur validation experience of a plasma-based NGS assay advances current knowledge about translating cfDNA testing into clinical practice and supports the application of plasma assays in the management of oncology patients with metastatic disease. With an in-house method that minimizes the need for invasive procedures, on-site cfDNA testing supplements tissue biopsy to guide precision therapy and is entitled to become a routine practice.Implications for practiceThis study proposes a solution for decentralized liquid biopsy testing based on validation of a next-generation sequencing (NGS) test that detects four classes of genomic alterations in blood: sequence mutations (single nucleotide substitutions or insertions and deletions), fusions, amplifications, and microsatellite instability (MSI). Although there are reference labs that perform single-site comprehensive liquid biopsy testing, the targeted assay this study validated can be established locally in any lab with capacity to offer clinical molecular pathology assays. To the authors' knowledge, this is the first report that validates evaluating an on-site plasma-based NGS test that detects the MSI status along with common sequence alterations encountered in solid tumors.

Dataset Information

Development and Validation of an Ultradeep Next-Generation Sequencing Assay for Testing of Plasma Cell-Free DNA from Patients with Advanced Cancer.

Publications

Development and Validation of an Ultradeep Next-Generation Sequencing Assay for Testing of Plasma Cell-Free DNA from Patients with Advanced Cancer.

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