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Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection
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 all...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer International Publishing
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675782/ https://www.ncbi.nlm.nih.gov/pubmed/31209714 http://dx.doi.org/10.1007/s40291-019-00406-0 |
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author | Atkins, Alexander Gupta, Pratyush Zhang, Bing Melody Tsai, Wen-Sy Lucas, Julian Javey, Manana Vora, Anagh Mei, Rui |
author_facet | Atkins, Alexander Gupta, Pratyush Zhang, Bing Melody Tsai, Wen-Sy Lucas, Julian Javey, Manana Vora, Anagh Mei, Rui |
author_sort | Atkins, Alexander |
collection | PubMed |
description | 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s40291-019-00406-0) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-6675782 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Springer International Publishing |
record_format | MEDLINE/PubMed |
spelling | pubmed-66757822019-08-14 Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection Atkins, Alexander Gupta, Pratyush Zhang, Bing Melody Tsai, Wen-Sy Lucas, Julian Javey, Manana Vora, Anagh Mei, Rui Mol Diagn Ther Original Research Article 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s40291-019-00406-0) contains supplementary material, which is available to authorized users. Springer International Publishing 2019-06-17 2019 /pmc/articles/PMC6675782/ /pubmed/31209714 http://dx.doi.org/10.1007/s40291-019-00406-0 Text en © The Author(s) 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommercial use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. |
spellingShingle | Original Research Article Atkins, Alexander Gupta, Pratyush Zhang, Bing Melody Tsai, Wen-Sy Lucas, Julian Javey, Manana Vora, Anagh Mei, Rui Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection |
title | Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection |
title_full | Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection |
title_fullStr | Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection |
title_full_unstemmed | Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection |
title_short | Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection |
title_sort | detection of circulating tumor dna with a single-molecule sequencing analysis validated for targeted and immunotherapy selection |
topic | Original Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6675782/ https://www.ncbi.nlm.nih.gov/pubmed/31209714 http://dx.doi.org/10.1007/s40291-019-00406-0 |
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