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Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection.
Mol Diagn Ther 2019; 23(4):521-535MD

Abstract

INTRODUCTION

Comprehensive genetic cancer profiling using circulating tumor DNA has enabled the detection of National Comprehensive Cancer Network (NCCN) guideline-recommended somatic alterations from a single, non-invasive blood draw. However, reliably detecting somatic variants at low variant allele fractions (VAFs) remains a challenge for next-generation sequencing (NGS)-based tests. We have developed the single-molecule sequencing (SMSEQ) platform to address these challenges.

METHODS

The OncoLBx assay utilizes the SMSEQ platform to optimize cell-free DNA extraction and library preparation with variant type-specific calling algorithms to improve sensitivity and specificity. OncoLBx is a pan-cancer panel for solid tumors targeting 75 genes and five microsatellite sites analyzing five classes of NCCN-recommended somatic variants: single-nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs), fusions and microsatellite instability (MSI). Circulating DNA was extracted from plasma, followed by library preparation using SMSEQ. Analytical validation was performed according to recently published American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines and established the limit of detection (LOD), sensitivity, specificity, accuracy and reproducibility using 126 gold-standard reference samples, healthy donor samples verified by whole-exome sequencing by an external College of American Pathologists (CAP) reference lab and cell lines with known variants. Results were analyzed using a locus-specific modeling algorithm.

RESULTS

We have demonstrated that OncoLBx detects VAFs of ≥ 0.1% for SNVs and indels, ≥ 0.5% for fusions, ≥ 4.5 copies for CNVs and ≥ 2% for MSI, with all variant types having specificity ≥ 99.999%. Diagnostic performance of paired samples displays 80% sensitivity and > 99.999% clinical specificity. Clinical utility and performance were assessed in 416 solid tumor samples. Variants were detected in 79% of samples, for which 87.34% of positive samples had available targeted therapy.

Authors+Show Affiliations

CellMax Life U.S., 1271 Oakmead Parkway, Sunnyvale, CA, 94085, USA.CellMax Life U.S., 1271 Oakmead Parkway, Sunnyvale, CA, 94085, USA.Department of Clinical Pathology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA, 94305, USA.Chang Gung Memorial Hospital, #5, Fuxing Street, Guishan District, Taoyuan, 333, Taiwan.CellMax Life U.S., 1271 Oakmead Parkway, Sunnyvale, CA, 94085, USA.CellMax Life U.S., 1271 Oakmead Parkway, Sunnyvale, CA, 94085, USA.CellMax Life U.S., 1271 Oakmead Parkway, Sunnyvale, CA, 94085, USA. Molecular Genetic Pathology Laboratory Services, St. Louis, MO, USA.CellMax Life U.S., 1271 Oakmead Parkway, Sunnyvale, CA, 94085, USA. rui@cellmaxlife.com.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31209714

Citation

Atkins, Alexander, et al. "Detection of Circulating Tumor DNA With a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection." Molecular Diagnosis & Therapy, vol. 23, no. 4, 2019, pp. 521-535.
Atkins A, Gupta P, Zhang BM, et al. Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection. Mol Diagn Ther. 2019;23(4):521-535.
Atkins, A., Gupta, P., Zhang, B. M., Tsai, W. S., Lucas, J., Javey, M., ... Mei, R. (2019). Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection. Molecular Diagnosis & Therapy, 23(4), pp. 521-535. doi:10.1007/s40291-019-00406-0.
Atkins A, et al. Detection of Circulating Tumor DNA With a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection. Mol Diagn Ther. 2019;23(4):521-535. PubMed PMID: 31209714.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection. AU - Atkins,Alexander, AU - Gupta,Pratyush, AU - Zhang,Bing Melody, AU - Tsai,Wen-Sy, AU - Lucas,Julian, AU - Javey,Manana, AU - Vora,Anagh, AU - Mei,Rui, PY - 2019/6/19/pubmed PY - 2019/6/19/medline PY - 2019/6/19/entrez SP - 521 EP - 535 JF - Molecular diagnosis & therapy JO - Mol Diagn Ther VL - 23 IS - 4 N2 - INTRODUCTION: Comprehensive genetic cancer profiling using circulating tumor DNA has enabled the detection of National Comprehensive Cancer Network (NCCN) guideline-recommended somatic alterations from a single, non-invasive blood draw. However, reliably detecting somatic variants at low variant allele fractions (VAFs) remains a challenge for next-generation sequencing (NGS)-based tests. We have developed the single-molecule sequencing (SMSEQ) platform to address these challenges. METHODS: The OncoLBx assay utilizes the SMSEQ platform to optimize cell-free DNA extraction and library preparation with variant type-specific calling algorithms to improve sensitivity and specificity. OncoLBx is a pan-cancer panel for solid tumors targeting 75 genes and five microsatellite sites analyzing five classes of NCCN-recommended somatic variants: single-nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs), fusions and microsatellite instability (MSI). Circulating DNA was extracted from plasma, followed by library preparation using SMSEQ. Analytical validation was performed according to recently published American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines and established the limit of detection (LOD), sensitivity, specificity, accuracy and reproducibility using 126 gold-standard reference samples, healthy donor samples verified by whole-exome sequencing by an external College of American Pathologists (CAP) reference lab and cell lines with known variants. Results were analyzed using a locus-specific modeling algorithm. RESULTS: We have demonstrated that OncoLBx detects VAFs of ≥ 0.1% for SNVs and indels, ≥ 0.5% for fusions, ≥ 4.5 copies for CNVs and ≥ 2% for MSI, with all variant types having specificity ≥ 99.999%. Diagnostic performance of paired samples displays 80% sensitivity and > 99.999% clinical specificity. Clinical utility and performance were assessed in 416 solid tumor samples. Variants were detected in 79% of samples, for which 87.34% of positive samples had available targeted therapy. SN - 1179-2000 UR - https://www.unboundmedicine.com/medline/citation/31209714/Detection_of_Circulating_Tumor_DNA_with_a_Single-Molecule_Sequencing_Analysis_Validated_for_Targeted_and_Immunotherapy_Selection L2 - https://dx.doi.org/10.1007/s40291-019-00406-0 DB - PRIME DP - Unbound Medicine ER -