Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling
Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Hi...
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creator | Plagnol, Vincent Woodhouse, Samuel Howarth, Karen Lensing, Stefanie Smith, Matt Epstein, Michael Madi, Mikidache Smalley, Sarah Leroy, Catherine Hinton, Jonathan de Kievit, Frank Musgrave-Brown, Esther Herd, Colin Baker-Neblett, Katherine Brennan, Will Dimitrov, Peter Campbell, Nathan Morris, Clive Rosenfeld, Nitzan Clark, James Gale, Davina Platt, Jamie Calaway, John Jones, Greg Forshew, Tim |
description | Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications. |
doi_str_mv | 10.1371/journal.pone.0193802 |
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Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0193802</identifier><identifier>PMID: 29543828</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Assaying ; Biology and Life Sciences ; Biopsy ; Cancer ; Carcinoma, Non-Small-Cell Lung - blood ; Carcinoma, Non-Small-Cell Lung - genetics ; Care and treatment ; Circulating Tumor DNA - blood ; Cohort Studies ; Deoxyribonucleic acid ; Diagnosis ; DNA ; DNA fingerprinting ; Epidermal growth factor receptors ; ErbB-2 protein ; Fibroblast growth factor receptor 1 ; Gene mutation ; Gene sequencing ; Genetic aspects ; High-throughput screening (Biochemical assaying) ; Humans ; Liquid Biopsy - methods ; Lung cancer ; Lung diseases ; Mathematical analysis ; Medical research ; Medicine and Health Sciences ; Methods ; Mutation ; Non-small cell lung carcinoma ; Operators ; Patients ; Polymerase Chain Reaction ; Profiling ; R&D ; Reproducibility of Results ; Research & development ; Sensitivity ; Sensitivity analysis ; Sensitivity and Specificity ; Sequence Analysis, DNA - methods ; Small cell lung cancer ; Therapeutic applications ; Tubes</subject><ispartof>PloS one, 2018-03, Vol.13 (3), p.e0193802-e0193802</ispartof><rights>COPYRIGHT 2018 Public Library of Science</rights><rights>2018 Plagnol et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications.</description><subject>Assaying</subject><subject>Biology and Life Sciences</subject><subject>Biopsy</subject><subject>Cancer</subject><subject>Carcinoma, Non-Small-Cell Lung - blood</subject><subject>Carcinoma, Non-Small-Cell Lung - genetics</subject><subject>Care and treatment</subject><subject>Circulating Tumor DNA - blood</subject><subject>Cohort Studies</subject><subject>Deoxyribonucleic acid</subject><subject>Diagnosis</subject><subject>DNA</subject><subject>DNA fingerprinting</subject><subject>Epidermal growth factor receptors</subject><subject>ErbB-2 protein</subject><subject>Fibroblast growth factor receptor 1</subject><subject>Gene mutation</subject><subject>Gene sequencing</subject><subject>Genetic aspects</subject><subject>High-throughput screening (Biochemical 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validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling</title><author>Plagnol, Vincent ; Woodhouse, Samuel ; Howarth, Karen ; Lensing, Stefanie ; Smith, Matt ; Epstein, Michael ; Madi, Mikidache ; Smalley, Sarah ; Leroy, Catherine ; Hinton, Jonathan ; de Kievit, Frank ; Musgrave-Brown, Esther ; Herd, Colin ; Baker-Neblett, Katherine ; Brennan, Will ; Dimitrov, Peter ; Campbell, Nathan ; Morris, Clive ; Rosenfeld, Nitzan ; Clark, James ; Gale, Davina ; Platt, Jamie ; Calaway, John ; Jones, Greg ; Forshew, Tim</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c719t-2ae553d10014c2c9d002a6a2ab3bc69419d343d6ea2a450c0fac68157412f2813</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>Assaying</topic><topic>Biology and Life Sciences</topic><topic>Biopsy</topic><topic>Cancer</topic><topic>Carcinoma, Non-Small-Cell Lung - 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Mikidache</au><au>Smalley, Sarah</au><au>Leroy, Catherine</au><au>Hinton, Jonathan</au><au>de Kievit, Frank</au><au>Musgrave-Brown, Esther</au><au>Herd, Colin</au><au>Baker-Neblett, Katherine</au><au>Brennan, Will</au><au>Dimitrov, Peter</au><au>Campbell, Nathan</au><au>Morris, Clive</au><au>Rosenfeld, Nitzan</au><au>Clark, James</au><au>Gale, Davina</au><au>Platt, Jamie</au><au>Calaway, John</au><au>Jones, Greg</au><au>Forshew, Tim</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2018-03-15</date><risdate>2018</risdate><volume>13</volume><issue>3</issue><spage>e0193802</spage><epage>e0193802</epage><pages>e0193802-e0193802</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>29543828</pmid><doi>10.1371/journal.pone.0193802</doi><tpages>e0193802</tpages><orcidid>https://orcid.org/0000-0003-2093-7020</orcidid><oa>free_for_read</oa></addata></record> |
fulltext | fulltext |
identifier | ISSN: 1932-6203 |
ispartof | PloS one, 2018-03, Vol.13 (3), p.e0193802-e0193802 |
issn | 1932-6203 1932-6203 |
language | eng |
recordid | cdi_plos_journals_2014447539 |
source | Public Library of Science (PLoS) Journals Open Access; MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry |
subjects | Assaying Biology and Life Sciences Biopsy Cancer Carcinoma, Non-Small-Cell Lung - blood Carcinoma, Non-Small-Cell Lung - genetics Care and treatment Circulating Tumor DNA - blood Cohort Studies Deoxyribonucleic acid Diagnosis DNA DNA fingerprinting Epidermal growth factor receptors ErbB-2 protein Fibroblast growth factor receptor 1 Gene mutation Gene sequencing Genetic aspects High-throughput screening (Biochemical assaying) Humans Liquid Biopsy - methods Lung cancer Lung diseases Mathematical analysis Medical research Medicine and Health Sciences Methods Mutation Non-small cell lung carcinoma Operators Patients Polymerase Chain Reaction Profiling R&D Reproducibility of Results Research & development Sensitivity Sensitivity analysis Sensitivity and Specificity Sequence Analysis, DNA - methods Small cell lung cancer Therapeutic applications Tubes |
title | Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling |
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