Validation of a Clinical-Grade Assay to Measure Donor-Derived Cell-Free DNA in Solid Organ Transplant Recipients
The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant...
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Veröffentlicht in: | The Journal of molecular diagnostics : JMD 2016-11, Vol.18 (6), p.890-902 |
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creator | Grskovic, Marica Hiller, David J Eubank, Lane A Sninsky, John J Christopherson, Cindy Collins, John P Thompson, Kathryn Song, Mindy Wang, Yue S Ross, David Nelles, Mitchell J Yee, James P Wilber, Judith C Crespo-Leiro, Maria G Scott, Susan L Woodward, Robert N |
description | The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance. |
doi_str_mv | 10.1016/j.jmoldx.2016.07.003 |
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However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance.</description><identifier>ISSN: 1525-1578</identifier><identifier>EISSN: 1943-7811</identifier><identifier>DOI: 10.1016/j.jmoldx.2016.07.003</identifier><identifier>PMID: 27727019</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Alleles ; Cell Line ; DNA - blood ; DNA - genetics ; Female ; Gene Frequency ; Genetic Markers ; Genetic Testing - methods ; Genetic Testing - standards ; Genotype ; Graft Rejection - diagnosis ; Graft Rejection - genetics ; Graft Rejection - immunology ; High-Throughput Nucleotide Sequencing ; Humans ; Male ; Organ Transplantation ; Pathology ; Polymorphism, Single Nucleotide ; Reference Standards ; Reproducibility of Results ; Sensitivity and Specificity ; Tissue Donors ; Transplant Recipients</subject><ispartof>The Journal of molecular diagnostics : JMD, 2016-11, Vol.18 (6), p.890-902</ispartof><rights>American Society for Investigative Pathology and the Association for Molecular Pathology</rights><rights>2016 American Society for Investigative Pathology and the Association for Molecular Pathology</rights><rights>Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c463t-cc7588bda30646673058b995c7f4980343be582af32da8891d4bf5ddc2c0c1ef3</citedby><cites>FETCH-LOGICAL-c463t-cc7588bda30646673058b995c7f4980343be582af32da8891d4bf5ddc2c0c1ef3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S1525157816301386$$EHTML$$P50$$Gelsevier$$Hfree_for_read</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27727019$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Grskovic, Marica</creatorcontrib><creatorcontrib>Hiller, David J</creatorcontrib><creatorcontrib>Eubank, Lane A</creatorcontrib><creatorcontrib>Sninsky, John J</creatorcontrib><creatorcontrib>Christopherson, Cindy</creatorcontrib><creatorcontrib>Collins, John P</creatorcontrib><creatorcontrib>Thompson, Kathryn</creatorcontrib><creatorcontrib>Song, Mindy</creatorcontrib><creatorcontrib>Wang, Yue S</creatorcontrib><creatorcontrib>Ross, David</creatorcontrib><creatorcontrib>Nelles, Mitchell J</creatorcontrib><creatorcontrib>Yee, James P</creatorcontrib><creatorcontrib>Wilber, Judith C</creatorcontrib><creatorcontrib>Crespo-Leiro, Maria G</creatorcontrib><creatorcontrib>Scott, Susan L</creatorcontrib><creatorcontrib>Woodward, Robert N</creatorcontrib><title>Validation of a Clinical-Grade Assay to Measure Donor-Derived Cell-Free DNA in Solid Organ Transplant Recipients</title><title>The Journal of molecular diagnostics : JMD</title><addtitle>J Mol Diagn</addtitle><description>The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance.</description><subject>Alleles</subject><subject>Cell Line</subject><subject>DNA - blood</subject><subject>DNA - genetics</subject><subject>Female</subject><subject>Gene Frequency</subject><subject>Genetic Markers</subject><subject>Genetic Testing - methods</subject><subject>Genetic Testing - standards</subject><subject>Genotype</subject><subject>Graft Rejection - diagnosis</subject><subject>Graft Rejection - genetics</subject><subject>Graft Rejection - immunology</subject><subject>High-Throughput Nucleotide Sequencing</subject><subject>Humans</subject><subject>Male</subject><subject>Organ Transplantation</subject><subject>Pathology</subject><subject>Polymorphism, Single Nucleotide</subject><subject>Reference Standards</subject><subject>Reproducibility of Results</subject><subject>Sensitivity and Specificity</subject><subject>Tissue Donors</subject><subject>Transplant Recipients</subject><issn>1525-1578</issn><issn>1943-7811</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUtv1DAUhS0EoqXwDxDykk2CH4ntbJBG0wdIhUq0sLUc-wY5ZOxgJxXz7_FoBhZsWPnaOvdc3-8g9JqSmhIq3o31uIuT-1WzcquJrAnhT9A57RpeSUXp01K3rK1oK9UZepHzSAhtGsGeozMmJZOEdudo_mYm78ziY8BxwAZvJx-8NVN1k4wDvMnZ7PES8ScweU2AL2OIqbqE5B_B4S1MU3WdoLx_3mAf8H0sdvgufTcBPyQT8jyZsOAvYP3sISz5JXo2mCnDq9N5gb5eXz1sP1S3dzcft5vbyjaCL5W1slWqd4YT0QghOWlV33WtlUPTKcIb3kOrmBk4c0apjrqmH1rnLLPEUhj4BXp79J1T_LlCXvTOZ1u-awLENWuqeMs7qURXpM1RalPMOcGg5-R3Ju01JfrAWo_6yFofWGsidWFd2t6cJqz9Dtzfpj9wi-D9UQBlz0cPSWdbGFhwPoFdtIv-fxP-NbCndH7AHvIY1xQKQ011Zpro-0Peh7ip4IRyJfhv712mPg</recordid><startdate>20161101</startdate><enddate>20161101</enddate><creator>Grskovic, Marica</creator><creator>Hiller, David J</creator><creator>Eubank, Lane A</creator><creator>Sninsky, John J</creator><creator>Christopherson, Cindy</creator><creator>Collins, John P</creator><creator>Thompson, Kathryn</creator><creator>Song, Mindy</creator><creator>Wang, Yue S</creator><creator>Ross, David</creator><creator>Nelles, Mitchell J</creator><creator>Yee, James P</creator><creator>Wilber, Judith C</creator><creator>Crespo-Leiro, Maria G</creator><creator>Scott, Susan L</creator><creator>Woodward, Robert N</creator><general>Elsevier Inc</general><scope>6I.</scope><scope>AAFTH</scope><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>7X8</scope></search><sort><creationdate>20161101</creationdate><title>Validation of a Clinical-Grade Assay to Measure Donor-Derived Cell-Free DNA in Solid Organ Transplant Recipients</title><author>Grskovic, Marica ; Hiller, David J ; Eubank, Lane A ; Sninsky, John J ; Christopherson, Cindy ; Collins, John P ; Thompson, Kathryn ; Song, Mindy ; Wang, Yue S ; Ross, David ; Nelles, Mitchell J ; Yee, James P ; Wilber, Judith C ; Crespo-Leiro, Maria G ; Scott, Susan L ; Woodward, Robert N</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c463t-cc7588bda30646673058b995c7f4980343be582af32da8891d4bf5ddc2c0c1ef3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>Alleles</topic><topic>Cell Line</topic><topic>DNA - blood</topic><topic>DNA - genetics</topic><topic>Female</topic><topic>Gene Frequency</topic><topic>Genetic Markers</topic><topic>Genetic Testing - methods</topic><topic>Genetic Testing - standards</topic><topic>Genotype</topic><topic>Graft Rejection - diagnosis</topic><topic>Graft Rejection - genetics</topic><topic>Graft Rejection - immunology</topic><topic>High-Throughput Nucleotide Sequencing</topic><topic>Humans</topic><topic>Male</topic><topic>Organ Transplantation</topic><topic>Pathology</topic><topic>Polymorphism, Single Nucleotide</topic><topic>Reference Standards</topic><topic>Reproducibility of Results</topic><topic>Sensitivity and Specificity</topic><topic>Tissue Donors</topic><topic>Transplant Recipients</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Grskovic, Marica</creatorcontrib><creatorcontrib>Hiller, David J</creatorcontrib><creatorcontrib>Eubank, Lane A</creatorcontrib><creatorcontrib>Sninsky, John J</creatorcontrib><creatorcontrib>Christopherson, Cindy</creatorcontrib><creatorcontrib>Collins, John P</creatorcontrib><creatorcontrib>Thompson, Kathryn</creatorcontrib><creatorcontrib>Song, Mindy</creatorcontrib><creatorcontrib>Wang, Yue S</creatorcontrib><creatorcontrib>Ross, David</creatorcontrib><creatorcontrib>Nelles, Mitchell J</creatorcontrib><creatorcontrib>Yee, James P</creatorcontrib><creatorcontrib>Wilber, Judith C</creatorcontrib><creatorcontrib>Crespo-Leiro, Maria G</creatorcontrib><creatorcontrib>Scott, Susan L</creatorcontrib><creatorcontrib>Woodward, Robert N</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>The Journal of molecular diagnostics : JMD</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Grskovic, Marica</au><au>Hiller, David J</au><au>Eubank, Lane A</au><au>Sninsky, John J</au><au>Christopherson, Cindy</au><au>Collins, John P</au><au>Thompson, Kathryn</au><au>Song, Mindy</au><au>Wang, Yue S</au><au>Ross, David</au><au>Nelles, Mitchell J</au><au>Yee, James P</au><au>Wilber, Judith C</au><au>Crespo-Leiro, Maria G</au><au>Scott, Susan L</au><au>Woodward, Robert N</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Validation of a Clinical-Grade Assay to Measure Donor-Derived Cell-Free DNA in Solid Organ Transplant Recipients</atitle><jtitle>The Journal of molecular diagnostics : JMD</jtitle><addtitle>J Mol Diagn</addtitle><date>2016-11-01</date><risdate>2016</risdate><volume>18</volume><issue>6</issue><spage>890</spage><epage>902</epage><pages>890-902</pages><issn>1525-1578</issn><eissn>1943-7811</eissn><abstract>The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>27727019</pmid><doi>10.1016/j.jmoldx.2016.07.003</doi><tpages>13</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Alleles Cell Line DNA - blood DNA - genetics Female Gene Frequency Genetic Markers Genetic Testing - methods Genetic Testing - standards Genotype Graft Rejection - diagnosis Graft Rejection - genetics Graft Rejection - immunology High-Throughput Nucleotide Sequencing Humans Male Organ Transplantation Pathology Polymorphism, Single Nucleotide Reference Standards Reproducibility of Results Sensitivity and Specificity Tissue Donors Transplant Recipients |
title | Validation of a Clinical-Grade Assay to Measure Donor-Derived Cell-Free DNA in Solid Organ Transplant Recipients |
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