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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

Frequency distribution of single nucleotide variant (SNV) mutant allele fractions (MAFs) for the first 1,000 consecutive patients tested in clinical practice (broad range of non-hematologic malignancies).On the left the three peaks represent kernel distribution density plots of the frequencies of somatic and germline mutant allele fractions. By their low concentrations, somatic mutations (far left-hand small peak) can be generally distinguished from heterozygous germline SNVs around 50% MAF (middle peak) and homozygous germline SNVs around 100% MAF (right-hand peak). Cell-free DNA is both leukocyte-derived and tumor-derived, with the germline DNA generally representing the bulk of the cfDNA. On the Guardant360 panel the molecular tumor board filters out the germline SNVs and only the somatic mutations are reported. The red curve in the right-hand figure shows the frequency distribution of the MAFs for the somatic SNVs only. The long tail reflects the rare patients with a somatic mutation at high MAF.
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pone.0140712.g006: Frequency distribution of single nucleotide variant (SNV) mutant allele fractions (MAFs) for the first 1,000 consecutive patients tested in clinical practice (broad range of non-hematologic malignancies).On the left the three peaks represent kernel distribution density plots of the frequencies of somatic and germline mutant allele fractions. By their low concentrations, somatic mutations (far left-hand small peak) can be generally distinguished from heterozygous germline SNVs around 50% MAF (middle peak) and homozygous germline SNVs around 100% MAF (right-hand peak). Cell-free DNA is both leukocyte-derived and tumor-derived, with the germline DNA generally representing the bulk of the cfDNA. On the Guardant360 panel the molecular tumor board filters out the germline SNVs and only the somatic mutations are reported. The red curve in the right-hand figure shows the frequency distribution of the MAFs for the somatic SNVs only. The long tail reflects the rare patients with a somatic mutation at high MAF.

Mentions: The frequency of distribution for germline SNPs and somatic SNVs is illustrated in Fig 6. The median (50th percentile) MAF for somatic mutations was 0.5%, with 1st quartile = 0.2%, 3rd quartile = 2.5% and maximum value = 71.5%. The long tail of higher MAFs resulted in a mean = 3.8%.


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)

Frequency distribution of single nucleotide variant (SNV) mutant allele fractions (MAFs) for the first 1,000 consecutive patients tested in clinical practice (broad range of non-hematologic malignancies).On the left the three peaks represent kernel distribution density plots of the frequencies of somatic and germline mutant allele fractions. By their low concentrations, somatic mutations (far left-hand small peak) can be generally distinguished from heterozygous germline SNVs around 50% MAF (middle peak) and homozygous germline SNVs around 100% MAF (right-hand peak). Cell-free DNA is both leukocyte-derived and tumor-derived, with the germline DNA generally representing the bulk of the cfDNA. On the Guardant360 panel the molecular tumor board filters out the germline SNVs and only the somatic mutations are reported. The red curve in the right-hand figure shows the frequency distribution of the MAFs for the somatic SNVs only. The long tail reflects the rare patients with a somatic mutation at high MAF.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140712.g006: Frequency distribution of single nucleotide variant (SNV) mutant allele fractions (MAFs) for the first 1,000 consecutive patients tested in clinical practice (broad range of non-hematologic malignancies).On the left the three peaks represent kernel distribution density plots of the frequencies of somatic and germline mutant allele fractions. By their low concentrations, somatic mutations (far left-hand small peak) can be generally distinguished from heterozygous germline SNVs around 50% MAF (middle peak) and homozygous germline SNVs around 100% MAF (right-hand peak). Cell-free DNA is both leukocyte-derived and tumor-derived, with the germline DNA generally representing the bulk of the cfDNA. On the Guardant360 panel the molecular tumor board filters out the germline SNVs and only the somatic mutations are reported. The red curve in the right-hand figure shows the frequency distribution of the MAFs for the somatic SNVs only. The long tail reflects the rare patients with a somatic mutation at high MAF.
Mentions: The frequency of distribution for germline SNPs and somatic SNVs is illustrated in Fig 6. The median (50th percentile) MAF for somatic mutations was 0.5%, with 1st quartile = 0.2%, 3rd quartile = 2.5% and maximum value = 71.5%. The long tail of higher MAFs resulted in a mean = 3.8%.

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