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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

Fig 2A illustrates output from Illumina HiSeq using standard library prep on cell-free DNA sample spiked with samples from ten cell lines with known single nucleotide variant (SNV) mutations. Germline single nucleotide polymorphisms (SNPs) (green dots) at either 50% (heterozygous) or 100% (homozygous) mutant allele fractions (MAF). In contrast, the ten somatic SNVs (red dots) are quantitated at much lower MAF typically encountered with cell-free circulating tumor DNA, and are obscured by the false positive “noise” associated with low DNA concentrations. The larger the targeted region, the more false positive signals are encountered. In this actual sample, sequencing the long targeted region (78 kbp) required for the 54-gene panel results in 224 false positives at the 0.1% to 10% MAFs, making accurate sequencing of ctDNA unworkable. Fig 2B utilizes the same sample as in Fig 2A but was analyzed with Digital Sequencing technology. Molecular techniques in the pre-analytic/pre-sequencing phase and bioinformatics in the post-sequencing phase are employed to eliminate the “noise” in a process analogous to the signal transduction processing-enabled conversion of analog voice and television signals to digital signals. The result is sensitivity to the level of 1–2 mutated DNA fragment molecules in up to 1,000 wild type (mostly leukocyte-derived) DNA fragments overlapping the same nucleotide base position, essentially eliminating the false positives normally encountered at low MAFs when sequencing large targeted regions.
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pone.0140712.g002: Fig 2A illustrates output from Illumina HiSeq using standard library prep on cell-free DNA sample spiked with samples from ten cell lines with known single nucleotide variant (SNV) mutations. Germline single nucleotide polymorphisms (SNPs) (green dots) at either 50% (heterozygous) or 100% (homozygous) mutant allele fractions (MAF). In contrast, the ten somatic SNVs (red dots) are quantitated at much lower MAF typically encountered with cell-free circulating tumor DNA, and are obscured by the false positive “noise” associated with low DNA concentrations. The larger the targeted region, the more false positive signals are encountered. In this actual sample, sequencing the long targeted region (78 kbp) required for the 54-gene panel results in 224 false positives at the 0.1% to 10% MAFs, making accurate sequencing of ctDNA unworkable. Fig 2B utilizes the same sample as in Fig 2A but was analyzed with Digital Sequencing technology. Molecular techniques in the pre-analytic/pre-sequencing phase and bioinformatics in the post-sequencing phase are employed to eliminate the “noise” in a process analogous to the signal transduction processing-enabled conversion of analog voice and television signals to digital signals. The result is sensitivity to the level of 1–2 mutated DNA fragment molecules in up to 1,000 wild type (mostly leukocyte-derived) DNA fragments overlapping the same nucleotide base position, essentially eliminating the false positives normally encountered at low MAFs when sequencing large targeted regions.

Mentions: We compared the performance of digital sequencing to traditional next-generation sequencing using TruSeq DNA Sample Preparation and bioinformatics pipelines with removal of low quality reads (i.e. Qscore < 30). When cancer cell-line cfDNA with ten known mutations are spiked at 0.1% MAFs into a background of cfDNA extracted from a healthy donor and sequenced with an Illumina HiSeq 2500 with standard prep, these SNVs are obscured by a plethora of false positives with MAFs of 0.05–5% (Fig 2A). Digital sequencing eliminates these false positives (as illustrated in Fig 2B) enabling high-quality sequencing of each single molecule of cfDNA. These figures also illustrate that both germline and tumor-derived SNVs are analyzed and quantitated simultaneously. SNV allele fractions around 50% or 100% generally (when tumor fraction in circulation is low) may be used to identify heterozygous or homozygous single nucleotide polymorphisms (SNPs), respectively.


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)

Fig 2A illustrates output from Illumina HiSeq using standard library prep on cell-free DNA sample spiked with samples from ten cell lines with known single nucleotide variant (SNV) mutations. Germline single nucleotide polymorphisms (SNPs) (green dots) at either 50% (heterozygous) or 100% (homozygous) mutant allele fractions (MAF). In contrast, the ten somatic SNVs (red dots) are quantitated at much lower MAF typically encountered with cell-free circulating tumor DNA, and are obscured by the false positive “noise” associated with low DNA concentrations. The larger the targeted region, the more false positive signals are encountered. In this actual sample, sequencing the long targeted region (78 kbp) required for the 54-gene panel results in 224 false positives at the 0.1% to 10% MAFs, making accurate sequencing of ctDNA unworkable. Fig 2B utilizes the same sample as in Fig 2A but was analyzed with Digital Sequencing technology. Molecular techniques in the pre-analytic/pre-sequencing phase and bioinformatics in the post-sequencing phase are employed to eliminate the “noise” in a process analogous to the signal transduction processing-enabled conversion of analog voice and television signals to digital signals. The result is sensitivity to the level of 1–2 mutated DNA fragment molecules in up to 1,000 wild type (mostly leukocyte-derived) DNA fragments overlapping the same nucleotide base position, essentially eliminating the false positives normally encountered at low MAFs when sequencing large targeted regions.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140712.g002: Fig 2A illustrates output from Illumina HiSeq using standard library prep on cell-free DNA sample spiked with samples from ten cell lines with known single nucleotide variant (SNV) mutations. Germline single nucleotide polymorphisms (SNPs) (green dots) at either 50% (heterozygous) or 100% (homozygous) mutant allele fractions (MAF). In contrast, the ten somatic SNVs (red dots) are quantitated at much lower MAF typically encountered with cell-free circulating tumor DNA, and are obscured by the false positive “noise” associated with low DNA concentrations. The larger the targeted region, the more false positive signals are encountered. In this actual sample, sequencing the long targeted region (78 kbp) required for the 54-gene panel results in 224 false positives at the 0.1% to 10% MAFs, making accurate sequencing of ctDNA unworkable. Fig 2B utilizes the same sample as in Fig 2A but was analyzed with Digital Sequencing technology. Molecular techniques in the pre-analytic/pre-sequencing phase and bioinformatics in the post-sequencing phase are employed to eliminate the “noise” in a process analogous to the signal transduction processing-enabled conversion of analog voice and television signals to digital signals. The result is sensitivity to the level of 1–2 mutated DNA fragment molecules in up to 1,000 wild type (mostly leukocyte-derived) DNA fragments overlapping the same nucleotide base position, essentially eliminating the false positives normally encountered at low MAFs when sequencing large targeted regions.
Mentions: We compared the performance of digital sequencing to traditional next-generation sequencing using TruSeq DNA Sample Preparation and bioinformatics pipelines with removal of low quality reads (i.e. Qscore < 30). When cancer cell-line cfDNA with ten known mutations are spiked at 0.1% MAFs into a background of cfDNA extracted from a healthy donor and sequenced with an Illumina HiSeq 2500 with standard prep, these SNVs are obscured by a plethora of false positives with MAFs of 0.05–5% (Fig 2A). Digital sequencing eliminates these false positives (as illustrated in Fig 2B) enabling high-quality sequencing of each single molecule of cfDNA. These figures also illustrate that both germline and tumor-derived SNVs are analyzed and quantitated simultaneously. SNV allele fractions around 50% or 100% generally (when tumor fraction in circulation is low) may be used to identify heterozygous or homozygous single nucleotide polymorphisms (SNPs), respectively.

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