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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

Analytic Specificity and Sensitivity, and Diagnostic Accuracy of the Guardant360 cell-free circulating tumor DNA assay.The matrix illustrates that a targeted region of 78 kbp was sequenced in each of 20 samples to identify single nucleotide variants at approximately 2.0–5.0% MAF (heterozygous) in circulating cfDNA. Green dots represent concordant single nucleotide variant (SNV) calls for digital sequencing of 10 ng cfDNA to calls made by whole exome sequencing of 2 μg of genomic (leukocyte) DNA from the same sample. The red dot represents the single false positive result in a cumulative 1.56 million bases sequenced. The far right-hand column illustrates the number of SNVs per sample identified with exome sequencing, and the penultimate column with digital sequencing, for a total of 365 and 366 SNVs, respectively.
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pone.0140712.g003: Analytic Specificity and Sensitivity, and Diagnostic Accuracy of the Guardant360 cell-free circulating tumor DNA assay.The matrix illustrates that a targeted region of 78 kbp was sequenced in each of 20 samples to identify single nucleotide variants at approximately 2.0–5.0% MAF (heterozygous) in circulating cfDNA. Green dots represent concordant single nucleotide variant (SNV) calls for digital sequencing of 10 ng cfDNA to calls made by whole exome sequencing of 2 μg of genomic (leukocyte) DNA from the same sample. The red dot represents the single false positive result in a cumulative 1.56 million bases sequenced. The far right-hand column illustrates the number of SNVs per sample identified with exome sequencing, and the penultimate column with digital sequencing, for a total of 365 and 366 SNVs, respectively.

Mentions: To assess the accuracy of our test, two micrograms of genomic DNA from 20 healthy and young donors (AllCells, Alameda, CA) were sent to an outside reference lab (Ambry Genetics, Aliso Viejo, CA) for whole exome sequencing. Ten nanograms of matched cfDNA samples were spiked at 5% (actual range observed was 4–10%, or 2–5% if for a heterozygous SNV) into another cfDNA sample and processed using Guardant360. Across the 78,000 base pair (78 kbp) panel, the whole-exome sequencing assay found 365 SNVs (mean 18 SNPs (range 12–27) per sample) and Guardant360 identified all 365 SNVs with one additional false positive SNV relative to whole exome sequencing (Fig 3). Thus analytic sensitivity for SNVs at 2% to 10% MAFs was 100%. The finding of 365 true positives and a single false positive in a cumulative targeted region of 1.56 million base pairs (20 samples x 78 kbp per sample), equates to analytic specificity of 99.9999%, or a 0.0001% false positive rate (Table 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)

Analytic Specificity and Sensitivity, and Diagnostic Accuracy of the Guardant360 cell-free circulating tumor DNA assay.The matrix illustrates that a targeted region of 78 kbp was sequenced in each of 20 samples to identify single nucleotide variants at approximately 2.0–5.0% MAF (heterozygous) in circulating cfDNA. Green dots represent concordant single nucleotide variant (SNV) calls for digital sequencing of 10 ng cfDNA to calls made by whole exome sequencing of 2 μg of genomic (leukocyte) DNA from the same sample. The red dot represents the single false positive result in a cumulative 1.56 million bases sequenced. The far right-hand column illustrates the number of SNVs per sample identified with exome sequencing, and the penultimate column with digital sequencing, for a total of 365 and 366 SNVs, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140712.g003: Analytic Specificity and Sensitivity, and Diagnostic Accuracy of the Guardant360 cell-free circulating tumor DNA assay.The matrix illustrates that a targeted region of 78 kbp was sequenced in each of 20 samples to identify single nucleotide variants at approximately 2.0–5.0% MAF (heterozygous) in circulating cfDNA. Green dots represent concordant single nucleotide variant (SNV) calls for digital sequencing of 10 ng cfDNA to calls made by whole exome sequencing of 2 μg of genomic (leukocyte) DNA from the same sample. The red dot represents the single false positive result in a cumulative 1.56 million bases sequenced. The far right-hand column illustrates the number of SNVs per sample identified with exome sequencing, and the penultimate column with digital sequencing, for a total of 365 and 366 SNVs, respectively.
Mentions: To assess the accuracy of our test, two micrograms of genomic DNA from 20 healthy and young donors (AllCells, Alameda, CA) were sent to an outside reference lab (Ambry Genetics, Aliso Viejo, CA) for whole exome sequencing. Ten nanograms of matched cfDNA samples were spiked at 5% (actual range observed was 4–10%, or 2–5% if for a heterozygous SNV) into another cfDNA sample and processed using Guardant360. Across the 78,000 base pair (78 kbp) panel, the whole-exome sequencing assay found 365 SNVs (mean 18 SNPs (range 12–27) per sample) and Guardant360 identified all 365 SNVs with one additional false positive SNV relative to whole exome sequencing (Fig 3). Thus analytic sensitivity for SNVs at 2% to 10% MAFs was 100%. The finding of 365 true positives and a single false positive in a cumulative targeted region of 1.56 million base pairs (20 samples x 78 kbp per sample), equates to analytic specificity of 99.9999%, or a 0.0001% false positive rate (Table 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