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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 7A is a comparison of tissue NGS results biopsied at five outside institutions compared to cfDNA sequencing at Guardant Health on 165 paired plasma samples from stage III-IV solid tumor cancer patients. Data summarizes diagnostic test performance for all 54 mutated tumor suppressor and oncogenes. The most commonly mutated genes were ALK, APC, BRAF, CDKN2A, CTNNB1, FBXW7, KRAS, NRAS, PIK3CA, PTEN, and TP53. Sensitivity, specificity and diagnostic accuracy are shown with 95% confidence intervals.Fig 7B illustrates the two by two contingency tables corresponding to Fig 7A. On the left cfDNA NGS results are compared to tissue-based NGS as the reference standard. On the right tissue-based NGS results are compared to cfDNA findings as the reference standard. All gene mutations found in cfDNA and tissue DNA based on NGS of 54 genes are shown in S2 Table. Both methods demonstrate similarly high sensitivity and near-perfect specificity. For cfDNA, sensitivity is limited by the amount of tumor DNA shed into circulation and for tissue, sensitivity is likely limited by sampling error related to intra-or inter-tumor heterogeneity. The sampling error on tissue samples may be related to sub-sampling of tumor heterogeneity by needle or surgical biopsy.
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pone.0140712.g007: Fig 7A is a comparison of tissue NGS results biopsied at five outside institutions compared to cfDNA sequencing at Guardant Health on 165 paired plasma samples from stage III-IV solid tumor cancer patients. Data summarizes diagnostic test performance for all 54 mutated tumor suppressor and oncogenes. The most commonly mutated genes were ALK, APC, BRAF, CDKN2A, CTNNB1, FBXW7, KRAS, NRAS, PIK3CA, PTEN, and TP53. Sensitivity, specificity and diagnostic accuracy are shown with 95% confidence intervals.Fig 7B illustrates the two by two contingency tables corresponding to Fig 7A. On the left cfDNA NGS results are compared to tissue-based NGS as the reference standard. On the right tissue-based NGS results are compared to cfDNA findings as the reference standard. All gene mutations found in cfDNA and tissue DNA based on NGS of 54 genes are shown in S2 Table. Both methods demonstrate similarly high sensitivity and near-perfect specificity. For cfDNA, sensitivity is limited by the amount of tumor DNA shed into circulation and for tissue, sensitivity is likely limited by sampling error related to intra-or inter-tumor heterogeneity. The sampling error on tissue samples may be related to sub-sampling of tumor heterogeneity by needle or surgical biopsy.

Mentions: A 165 patient subset of the 510 samples from the five centers had matched plasma and tissue samples. Solid tumor cancer histologies included 57 colorectal cancers (CRC), 22 other GI (non-CRC), 18 melanoma, 18 lung cancer, 15 breast cancer, 8 genitourinary cancer, and 27 other cancer types of lesser frequency. 24% were stage III, 73% stage IV and 3% were unknown. The median age was 58 (range 33–88) and 57% were male and 43% female. Clinical sensitivity for digital sequencing for all mutated oncogenes compared to tissue-based NGS was 85.0%, specificity 99.6%, diagnostic accuracy 99.3%. For tissue-based NGS compared to digital sequencing of cfDNA as the reference, clinical sensitivity was 80.7%, specificity 99.7%, diagnostic accuracy 99.3% (Fig 7A and 7B). Both sample types were processed on an Illumina HiSeq 2500 next generation sequencer.


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 7A is a comparison of tissue NGS results biopsied at five outside institutions compared to cfDNA sequencing at Guardant Health on 165 paired plasma samples from stage III-IV solid tumor cancer patients. Data summarizes diagnostic test performance for all 54 mutated tumor suppressor and oncogenes. The most commonly mutated genes were ALK, APC, BRAF, CDKN2A, CTNNB1, FBXW7, KRAS, NRAS, PIK3CA, PTEN, and TP53. Sensitivity, specificity and diagnostic accuracy are shown with 95% confidence intervals.Fig 7B illustrates the two by two contingency tables corresponding to Fig 7A. On the left cfDNA NGS results are compared to tissue-based NGS as the reference standard. On the right tissue-based NGS results are compared to cfDNA findings as the reference standard. All gene mutations found in cfDNA and tissue DNA based on NGS of 54 genes are shown in S2 Table. Both methods demonstrate similarly high sensitivity and near-perfect specificity. For cfDNA, sensitivity is limited by the amount of tumor DNA shed into circulation and for tissue, sensitivity is likely limited by sampling error related to intra-or inter-tumor heterogeneity. The sampling error on tissue samples may be related to sub-sampling of tumor heterogeneity by needle or surgical biopsy.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608804&req=5

pone.0140712.g007: Fig 7A is a comparison of tissue NGS results biopsied at five outside institutions compared to cfDNA sequencing at Guardant Health on 165 paired plasma samples from stage III-IV solid tumor cancer patients. Data summarizes diagnostic test performance for all 54 mutated tumor suppressor and oncogenes. The most commonly mutated genes were ALK, APC, BRAF, CDKN2A, CTNNB1, FBXW7, KRAS, NRAS, PIK3CA, PTEN, and TP53. Sensitivity, specificity and diagnostic accuracy are shown with 95% confidence intervals.Fig 7B illustrates the two by two contingency tables corresponding to Fig 7A. On the left cfDNA NGS results are compared to tissue-based NGS as the reference standard. On the right tissue-based NGS results are compared to cfDNA findings as the reference standard. All gene mutations found in cfDNA and tissue DNA based on NGS of 54 genes are shown in S2 Table. Both methods demonstrate similarly high sensitivity and near-perfect specificity. For cfDNA, sensitivity is limited by the amount of tumor DNA shed into circulation and for tissue, sensitivity is likely limited by sampling error related to intra-or inter-tumor heterogeneity. The sampling error on tissue samples may be related to sub-sampling of tumor heterogeneity by needle or surgical biopsy.
Mentions: A 165 patient subset of the 510 samples from the five centers had matched plasma and tissue samples. Solid tumor cancer histologies included 57 colorectal cancers (CRC), 22 other GI (non-CRC), 18 melanoma, 18 lung cancer, 15 breast cancer, 8 genitourinary cancer, and 27 other cancer types of lesser frequency. 24% were stage III, 73% stage IV and 3% were unknown. The median age was 58 (range 33–88) and 57% were male and 43% female. Clinical sensitivity for digital sequencing for all mutated oncogenes compared to tissue-based NGS was 85.0%, specificity 99.6%, diagnostic accuracy 99.3%. For tissue-based NGS compared to digital sequencing of cfDNA as the reference, clinical sensitivity was 80.7%, specificity 99.7%, diagnostic accuracy 99.3% (Fig 7A and 7B). Both sample types were processed on an Illumina HiSeq 2500 next generation sequencer.

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