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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 Sequencingâ„¢ 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

Analytic Sensitivity of the Guardant360 digital sequencing method.Twenty nine SNV samples were diluted serially until they could no longer be measured with the assay. Each column represents successively greater serial dilutions of a given sample. The limit of detection (LOD) was 0.25% mutant allele frequency or fraction) (MAF), defined as the percentage at which > 80% of samples were detected. Note that almost 30% of samples were additionally detected at 0.1% MAF or lower, where 0.1% represents a single mutated DNA fragment out of 999 wild-type (leukocyte-derived) DNA fragments overlapping the same nucleotide base.
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pone.0140712.g004: Analytic Sensitivity of the Guardant360 digital sequencing method.Twenty nine SNV samples were diluted serially until they could no longer be measured with the assay. Each column represents successively greater serial dilutions of a given sample. The limit of detection (LOD) was 0.25% mutant allele frequency or fraction) (MAF), defined as the percentage at which > 80% of samples were detected. Note that almost 30% of samples were additionally detected at 0.1% MAF or lower, where 0.1% represents a single mutated DNA fragment out of 999 wild-type (leukocyte-derived) DNA fragments overlapping the same nucleotide base.

Mentions: Analytic sensitivity based on serial dilutions of 29 different SNVs was conducted in triplicate. The SNV fraction at which > 80% of SNVs are detected is defined as the limit of detection and was 0.25% (Fig 4). In 28% of the samples, dilution to 0.1% or lower could still be detected. This corresponds to one DNA fragment with a SNV at a given nucleotide base position divided by 999 wild-type DNA fragments overlapping the same base position.


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)

Analytic Sensitivity of the Guardant360 digital sequencing method.Twenty nine SNV samples were diluted serially until they could no longer be measured with the assay. Each column represents successively greater serial dilutions of a given sample. The limit of detection (LOD) was 0.25% mutant allele frequency or fraction) (MAF), defined as the percentage at which > 80% of samples were detected. Note that almost 30% of samples were additionally detected at 0.1% MAF or lower, where 0.1% represents a single mutated DNA fragment out of 999 wild-type (leukocyte-derived) DNA fragments overlapping the same nucleotide base.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140712.g004: Analytic Sensitivity of the Guardant360 digital sequencing method.Twenty nine SNV samples were diluted serially until they could no longer be measured with the assay. Each column represents successively greater serial dilutions of a given sample. The limit of detection (LOD) was 0.25% mutant allele frequency or fraction) (MAF), defined as the percentage at which > 80% of samples were detected. Note that almost 30% of samples were additionally detected at 0.1% MAF or lower, where 0.1% represents a single mutated DNA fragment out of 999 wild-type (leukocyte-derived) DNA fragments overlapping the same nucleotide base.
Mentions: Analytic sensitivity based on serial dilutions of 29 different SNVs was conducted in triplicate. The SNV fraction at which > 80% of SNVs are detected is defined as the limit of detection and was 0.25% (Fig 4). In 28% of the samples, dilution to 0.1% or lower could still be detected. This corresponds to one DNA fragment with a SNV at a given nucleotide base position divided by 999 wild-type DNA fragments overlapping the same base position.

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 Sequencingâ„¢ 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