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Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA.

Lanman RB, Mortimer SA, Zill OA, Sebisanovic D, Lopez R, Blau S, Collisson EA, Divers SG, Hoon DS, Kopetz ES, Lee J, Nikolinakos PG, Baca AM, Kermani BG, Eltoukhy H, Talasaz A - PLoS ONE (2015)

Bottom Line: Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%.Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue.The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Affairs, Guardant Health, Inc., Redwood City, California, United States of America.

ABSTRACT
Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital Sequencing™ is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient's cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.

No MeSH data available.


Related in: MedlinePlus

Workflow for the Guardant360 cell-free circulating DNA NGS genomic profile.(a) cfDNA is extracted from a routine blood draw. (b) 5.0–30 ng of DNA undergoes digital library preparation including oligonucleotide barcoding of each strand in each individual DNA fragment. Complete sequencing of 512 exons in 54 cancer-related genes is conducted with the HiSeq 2500 (Illumina). Multi-analyte algorithms and bioinformatics are used to reconstruct the progenitor cfDNA fragment sequences without false positives. (c) Sequence data are processed using a customized analysis pipeline designed to accurately detect the four major classes of genomic alterations. (d) Mutant allele fractions are reported quantitatively for somatic single nucleotide variants of clinical significance and distinguished from germline single nucleotide variants (SNVs) by reference to the COSMIC and dbSNP databases, as well as their concentrations.
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pone.0140712.g001: Workflow for the Guardant360 cell-free circulating DNA NGS genomic profile.(a) cfDNA is extracted from a routine blood draw. (b) 5.0–30 ng of DNA undergoes digital library preparation including oligonucleotide barcoding of each strand in each individual DNA fragment. Complete sequencing of 512 exons in 54 cancer-related genes is conducted with the HiSeq 2500 (Illumina). Multi-analyte algorithms and bioinformatics are used to reconstruct the progenitor cfDNA fragment sequences without false positives. (c) Sequence data are processed using a customized analysis pipeline designed to accurately detect the four major classes of genomic alterations. (d) Mutant allele fractions are reported quantitatively for somatic single nucleotide variants of clinical significance and distinguished from germline single nucleotide variants (SNVs) by reference to the COSMIC and dbSNP databases, as well as their concentrations.

Mentions: The Guardant360 cfDNA assay workflow is illustrated in Fig 1.


Analytical and Clinical Validation of a Digital Sequencing Panel for Quantitative, Highly Accurate Evaluation of Cell-Free Circulating Tumor DNA.

Lanman RB, Mortimer SA, Zill OA, Sebisanovic D, Lopez R, Blau S, Collisson EA, Divers SG, Hoon DS, Kopetz ES, Lee J, Nikolinakos PG, Baca AM, Kermani BG, Eltoukhy H, Talasaz A - PLoS ONE (2015)

Workflow for the Guardant360 cell-free circulating DNA NGS genomic profile.(a) cfDNA is extracted from a routine blood draw. (b) 5.0–30 ng of DNA undergoes digital library preparation including oligonucleotide barcoding of each strand in each individual DNA fragment. Complete sequencing of 512 exons in 54 cancer-related genes is conducted with the HiSeq 2500 (Illumina). Multi-analyte algorithms and bioinformatics are used to reconstruct the progenitor cfDNA fragment sequences without false positives. (c) Sequence data are processed using a customized analysis pipeline designed to accurately detect the four major classes of genomic alterations. (d) Mutant allele fractions are reported quantitatively for somatic single nucleotide variants of clinical significance and distinguished from germline single nucleotide variants (SNVs) by reference to the COSMIC and dbSNP databases, as well as their concentrations.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608804&req=5

pone.0140712.g001: Workflow for the Guardant360 cell-free circulating DNA NGS genomic profile.(a) cfDNA is extracted from a routine blood draw. (b) 5.0–30 ng of DNA undergoes digital library preparation including oligonucleotide barcoding of each strand in each individual DNA fragment. Complete sequencing of 512 exons in 54 cancer-related genes is conducted with the HiSeq 2500 (Illumina). Multi-analyte algorithms and bioinformatics are used to reconstruct the progenitor cfDNA fragment sequences without false positives. (c) Sequence data are processed using a customized analysis pipeline designed to accurately detect the four major classes of genomic alterations. (d) Mutant allele fractions are reported quantitatively for somatic single nucleotide variants of clinical significance and distinguished from germline single nucleotide variants (SNVs) by reference to the COSMIC and dbSNP databases, as well as their concentrations.
Mentions: The Guardant360 cfDNA assay workflow is illustrated in Fig 1.

Bottom Line: Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%.Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue.The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Affairs, Guardant Health, Inc., Redwood City, California, United States of America.

ABSTRACT
Next-generation sequencing of cell-free circulating solid tumor DNA addresses two challenges in contemporary cancer care. First this method of massively parallel and deep sequencing enables assessment of a comprehensive panel of genomic targets from a single sample, and second, it obviates the need for repeat invasive tissue biopsies. Digital Sequencing™ is a novel method for high-quality sequencing of circulating tumor DNA simultaneously across a comprehensive panel of over 50 cancer-related genes with a simple blood test. Here we report the analytic and clinical validation of the gene panel. Analytic sensitivity down to 0.1% mutant allele fraction is demonstrated via serial dilution studies of known samples. Near-perfect analytic specificity (> 99.9999%) enables complete coverage of many genes without the false positives typically seen with traditional sequencing assays at mutant allele frequencies or fractions below 5%. We compared digital sequencing of plasma-derived cell-free DNA to tissue-based sequencing on 165 consecutive matched samples from five outside centers in patients with stage III-IV solid tumor cancers. Clinical sensitivity of plasma-derived NGS was 85.0%, comparable to 80.7% sensitivity for tissue. The assay success rate on 1,000 consecutive samples in clinical practice was 99.8%. Digital sequencing of plasma-derived DNA is indicated in advanced cancer patients to prevent repeated invasive biopsies when the initial biopsy is inadequate, unobtainable for genomic testing, or uninformative, or when the patient's cancer has progressed despite treatment. Its clinical utility is derived from reduction in the costs, complications and delays associated with invasive tissue biopsies for genomic testing.

No MeSH data available.


Related in: MedlinePlus