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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Veröffentlicht in:PloS one 2018-03, Vol.13 (3), p.e0193802-e0193802
Hauptverfasser: 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
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container_issue 3
container_start_page e0193802
container_title PloS one
container_volume 13
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. 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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. 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development</subject><subject>Sensitivity</subject><subject>Sensitivity analysis</subject><subject>Sensitivity and Specificity</subject><subject>Sequence Analysis, DNA - methods</subject><subject>Small cell lung cancer</subject><subject>Therapeutic applications</subject><subject>Tubes</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><sourceid>DOA</sourceid><recordid>eNqNkl2LEzEUhgdR3LX6D0QDguhFaz6nMzdCWfwoLCz4dRtOk8w0JU26yUzZ_nszdnZpxQuvEk6evCfnzVsULwmeETYnHzahjx7cbBe8mWFSswrTR8Vl3tBpSTF7fLK_KJ6ltMFYsKosnxYXtBacVbS6LO4WWePQWQUO7cFZDZ0NHoUGAfLmrkOt8SYei8nc9sYr61vk7G1vNVrZsEsHBCnBATUhorVt15nzyXZ2b7sDWsUAGm2DM6p3ENEuhsa6LPG8eNKAS-bFuE6Kn58__bj6Or2--bK8WlxP1ZzU3ZSCEYJpgjHhiqpaY0yhBAortlJlzUmtGWe6NLnEBVa4AVVWRMw5oQ2tCJsUr4-6OxeSHE1LkmY9zueC1ZlYHgkdYCN30W4hHmQAK_8UQmwlxOyQM7ImjODaKADdcNrwSihGDKalrjQoYFnr49itX22NVsZ3EdyZ6PmJt2vZhr0UVf4ROjz33SgQQ3Y7dXJrkzLOgTehP767FhRnelK8-Qv993Qj1UIewPom5L5qEJULwQihc8JFpt6fUSr4Lv9-C31Kcvn92_-zN7_O2bcn7NqA69YpuH7IUzoH-RFUMaQUTfPgGcFyyPv9cHLIuxzznq-9OvX74dJ9wNlvh9v8vQ</recordid><startdate>20180315</startdate><enddate>20180315</enddate><creator>Plagnol, Vincent</creator><creator>Woodhouse, Samuel</creator><creator>Howarth, Karen</creator><creator>Lensing, Stefanie</creator><creator>Smith, Matt</creator><creator>Epstein, Michael</creator><creator>Madi, Mikidache</creator><creator>Smalley, Sarah</creator><creator>Leroy, Catherine</creator><creator>Hinton, Jonathan</creator><creator>de Kievit, Frank</creator><creator>Musgrave-Brown, Esther</creator><creator>Herd, Colin</creator><creator>Baker-Neblett, Katherine</creator><creator>Brennan, Will</creator><creator>Dimitrov, Peter</creator><creator>Campbell, Nathan</creator><creator>Morris, Clive</creator><creator>Rosenfeld, Nitzan</creator><creator>Clark, James</creator><creator>Gale, Davina</creator><creator>Platt, Jamie</creator><creator>Calaway, John</creator><creator>Jones, Greg</creator><creator>Forshew, Tim</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PHGZM</scope><scope>PHGZT</scope><scope>PIMPY</scope><scope>PJZUB</scope><scope>PKEHL</scope><scope>PPXIY</scope><scope>PQEST</scope><scope>PQGLB</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0003-2093-7020</orcidid></search><sort><creationdate>20180315</creationdate><title>Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling</title><author>Plagnol, Vincent ; 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Medical Research Collection</collection><collection>ProQuest One Academic Middle East (New)</collection><collection>ProQuest One Health &amp; Nursing</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Applied &amp; Life Sciences</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Engineering Collection</collection><collection>Environmental Science Collection</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Plagnol, Vincent</au><au>Woodhouse, Samuel</au><au>Howarth, Karen</au><au>Lensing, Stefanie</au><au>Smith, Matt</au><au>Epstein, Michael</au><au>Madi, 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>
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identifier ISSN: 1932-6203
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1932-6203
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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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