Development And Validation Of An Ngs-Based Assay To Detect All Classes Of Genomic Alterations In Circulating Tumor Cells (Ctcs) From Patients With Solid Tumors
CANCER RESEARCH(2015)
Abstract
Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PAPurposeWith increased use of targeted therapeutics, the ability to perform a ‘liquid biopsy’ on circulating tumor cells (CTCs) holds potential to address the need for a more representative and longitudinal means to monitor tumor burden in patients. However, the analytic and clinical validity of such an assay has proved difficult to establish, as the cell population in circulation is complex, and the CTC portion is heterogeneous, evolving, and highly dependent on clinical factors as well as CTC isolation methods. No studies to date have characterized CTCs for the full range of relevant genomic alterations in the context of patient-matched primary and metastatic tissue biopsies. We set out to develop a method to perform comprehensive NGS-based genomic profiling on phenotypically-isolated CTCs, and to compare CTC analysis with a comprehensive tissue-based assessment of paired primary and metastatic tissue in patients.MethodsAn integrated platform (VERSA) was employed for capture of EpCAM CTCs, enumeration and DNA extraction. Extracted DNA was amplified using a modified version of MDA-based whole genome amplification (WGA), followed by standard adaptor-ligated library construction and solution hybridization using a custom DNA baitset before sequencing on Illumina HiSeq. Data was analyzed using a computational workflow developed to detect base substitutions, indels, high-level copy number alterations, and select gene rearrangements. We performed extensive analytic validation of the assay using over 65 cell line mixtures, constructed to contain all classes of known variants across all allele frequencies (0-100%). We used the assay to analyze CTC DNA from 20 lung cancer patients, paired with analysis of temporally-matched primary and/or metastatic FFPE samples analyzed by FoundationOne, an FFPE tissue-based assay.ResultsValidation experiments showed that for samples meeting minimum acceptance criteria (50 cells at 50% purity), assay sensitivity exceeded 95% for detections of base substitutions, 85% for indels and rearrangements, and 80% for high-level copy number alterations, while maintaining tight specificity control. WGA-induced coverage bias limited sensitivity to low-level copy number alterations. Comparison of driver alterations across CTC samples and paired primary/metastatic tissue for 20 patients with lung cancer is ongoing.ConclusionsA comprehensive NGS-based approach to genomic profiling is feasible on CTCs isolated from lung cancer patients, and demonstrated accuracy sufficient to characterize actionable targets for clinical decision-making. However, more extensive tumor-type specific studies with a similar matched-tissue approach must be performed in order to establish full analytic and clinical validation, and assay specifications will need to be optimized to enable use on broader patient populations.Citation Format: Allison Welsh, Garrett M. Frampton, Zachary R. Chalmers, Jamie Sperger, Roman Yelensky, Doron Lipson, Geoff Otto, Lindsay Strotman, Scott Berry, Hannah Pezzi, Anne Traynor, David J. Beebe, Vincent A. Miller, Joshua M. Lang, Philip J. Stephens. Development and validation of an NGS-based assay to detect all classes of genomic alterations in circulating tumor cells (CTCs) from patients with solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1602. doi:10.1158/1538-7445.AM2015-1602
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Key words
tumor cells,solid tumors,genomic alterations,ngs-based
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