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Serial Next-Generation Sequencing of Circulating Cell-Free DNA Evaluating Tumor Clone Response To Molecularly Targeted Drug Administration.

ABSTRACT: PURPOSE:We evaluated whether next-generation sequencing (NGS) of circulating cell-free DNA (cfDNA) could be used for patient selection and as a tumor clone response biomarker in patients with advanced cancers participating in early-phase clinical trials of targeted drugs. EXPERIMENTAL DESIGN:Plasma samples from patients with known tumor mutations who completed at least two courses of investigational targeted therapy were collected monthly, until disease progression. NGS was performed sequentially on the Ion Torrent PGM platform. RESULTS:cfDNA was extracted from 39 patients with various tumor types. Treatments administered targeted mainly the PI3K-AKT-mTOR pathway (n = 28) or MEK (n = 7). Overall, 159 plasma samples were sequenced with a mean sequencing coverage achieved of 1,685X across experiments. At trial initiation (C1D1), 23 of 39 (59%) patients had at least one mutation identified in cfDNA (mean 2, range 1-5). Out of the 44 mutations identified at C1D1, TP53, PIK3CA and KRAS were the top 3 mutated genes identified, with 18 (41%), 9 (20%), 8 (18%) different mutations, respectively. Out of these 23 patients, 13 received a targeted drug matching their tumor profile. For the 23 patients with cfDNA mutation at C1D1, the monitoring of mutation allele frequency (AF) in consecutive plasma samples during treatment with targeted drugs demonstrated potential treatment associated clonal responses. Longitudinal monitoring of cfDNA samples with multiple mutations indicated the presence of separate clones behaving discordantly. Molecular changes at cfDNA mutation level were associated with time to disease progression by RECIST criteria. CONCLUSIONS:Targeted NGS of cfDNA has potential clinical utility to monitor the delivery of targeted therapies.

SUBMITTER: Frenel JS

PROVIDER: S-EPMC4580992 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Serial Next-Generation Sequencing of Circulating Cell-Free DNA Evaluating Tumor Clone Response To Molecularly Targeted Drug Administration.

Frenel Jean Sebastien JS Carreira Suzanne S Goodall Jane J Roda Desam D Perez-Lopez Raquel R Tunariu Nina N Riisnaes Ruth R Miranda Susana S Figueiredo Ines I Nava-Rodrigues Daniel D Smith Alan A Leux Christophe C Garcia-Murillas Isaac I Ferraldeschi Roberta R Lorente David D Mateo Joaquin J Ong Michael M Yap Timothy A TA Banerji Udai U Gasi Tandefelt Delila D Turner Nick N Attard Gerhardt G de Bono Johann S JS

Clinical cancer research : an official journal of the American Association for Cancer Research 20150617 20

<h4>Purpose</h4>We evaluated whether next-generation sequencing (NGS) of circulating cell-free DNA (cfDNA) could be used for patient selection and as a tumor clone response biomarker in patients with advanced cancers participating in early-phase clinical trials of targeted drugs.<h4>Experimental design</h4>Plasma samples from patients with known tumor mutations who completed at least two courses of investigational targeted therapy were collected monthly, until disease progression. NGS was perfor ...[more]

PMID: 26085511

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Project description:Currently available methods for the laboratory investigation of Legionella pneumophila outbreaks require organism culture. The ability to sequence L. pneumophila directly from clinical samples would significantly reduce delays. Here, we develop a method for targeted next-generation sequencing (NGS) of selected L. pneumophila genes utilizing a CRISPR/Cas9-based target enrichment system. We determine the method's utility by typing cultured L. pneumophila isolates and subsequently apply the method directly to patient samples. We sequenced 10 L. pneumophila isolates by 2 methods, (i) whole-genome sequencing (WGS) and (ii) targeted (CRISPR/Cas9-based) finding low-abundance sequences by hybridization (FLASH)-NGS, sequencing 57 selected genes. The targeted NGS of 57 genes was more efficient than WGS, and phylogenetic analysis of the 57 genes yielded the same classification of the L. pneumophila isolates as that based on analysis of whole-genome data. Furthermore, targeted NGS of L. pneumophila performed directly on patient respiratory samples correctly classified the patients according to their corresponding cultured isolates. This provides proof of concept that targeted NGS can be used to sequence L. pneumophila directly from patient samples. Studies on a larger number of patient samples will further validate this method. Nonetheless, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms. IMPORTANCE The bacterium Legionella pneumophila is responsible for outbreaks of serious and life-threatening pneumonia called Legionnaires' disease. There is a need for new molecular methods that allow investigation of Legionella outbreaks directly from patient samples, without the need for prior microbiological culture, which causes delays. Our study aims to address this problem. We have utilized a CRISPR/Cas9-based targeted next-generation sequencing (NGS) method that can be applied directly on human specimens. Furthermore, we show that analysis of the sequences of a small number of targeted genes offers the same classification of L. pneumophila as that based on data derived from the whole genome. Given the rising interest globally in sequencing pathogens directly from human samples, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms.

Dataset Information

Serial Next-Generation Sequencing of Circulating Cell-Free DNA Evaluating Tumor Clone Response To Molecularly Targeted Drug Administration.

Publications

Serial Next-Generation Sequencing of Circulating Cell-Free DNA Evaluating Tumor Clone Response To Molecularly Targeted Drug Administration.

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