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Fit-for-purpose quantitative liquid biopsy based droplet digital PCR assay development for detection of programmed cell death ligand-1 (PD-L1) RNA expression in PAXgene blood samples
Development of a clinically applicable liquid biopsy-based test for PD-L1 mRNA expression would be beneficial in providing complementary evidence to current immunohistochemistry assays. Hence, we report the development of a fit-for-purpose assay for detection of blood PD-L1 mRNA expression using dro...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8109819/ https://www.ncbi.nlm.nih.gov/pubmed/33970922 http://dx.doi.org/10.1371/journal.pone.0250849 |
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author | O’Rourke, Dennis Wang, Danyi Sanchez-Garcia, Jorge F. Cusano, Maria Perella Miller, Waldemar Cai, Ti Scheuenpflug, Juergen Feng, Zheng |
author_facet | O’Rourke, Dennis Wang, Danyi Sanchez-Garcia, Jorge F. Cusano, Maria Perella Miller, Waldemar Cai, Ti Scheuenpflug, Juergen Feng, Zheng |
author_sort | O’Rourke, Dennis |
collection | PubMed |
description | Development of a clinically applicable liquid biopsy-based test for PD-L1 mRNA expression would be beneficial in providing complementary evidence to current immunohistochemistry assays. Hence, we report the development of a fit-for-purpose assay for detection of blood PD-L1 mRNA expression using droplet digital polymerase chain reaction (ddPCR). TaqMan® assays were selected based on coverage of the PD-L1 gene and were tested for linearity and efficiency using real-time quantitative PCR. Four reference genes were analyzed in positive control cell line (A549 treated with interferon gamma, [IFN γ]) genomic DNA. The PD-L1 primer/probe sets were also evaluated in ddPCR for limit of blank, limit of detection, and precision. Finally, thirty-five healthy volunteer samples were evaluated to establish a baseline level of PD-L1 expression. In ddPCR, the limit of blank was determined to be 0 copies and the limit of detection was determined to be less than or equal to 19 copies of PD-L1. The average intra-run coefficient of variation in the ddPCR assay was 7.44% and average inter-run CV was 7.70%. Treatment of A549 cells with IFN γ resulted in a 6.7-fold increase in PD-L1 expression (21,580 copies in untreated cDNA versus 145,000 copies in treated cDNA). Analysis of healthy human samples yielded a median value of 1659 PD-L1 copies/μL with a range of 768–7510 copies/μL. The assay was transferred to an external service provider and results from our in-house experiments and those conducted externally shows a correlation of 0.994. In conclusion, a fit-for-purpose liquid biopsy-based, purely quantitative ddPCR assay for the detection of PD-L1 mRNA expression was developed and validated using PAXgene RNA blood samples. Linearity, reproducibility, limit of blank and limit of detection were measured and deemed suitable for clinical application. This ultra-sensitive liquid biopsy ddPCR assay has promising clinical potential in screening, longitudinal monitoring and disease progression detection. |
format | Online Article Text |
id | pubmed-8109819 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-81098192021-05-21 Fit-for-purpose quantitative liquid biopsy based droplet digital PCR assay development for detection of programmed cell death ligand-1 (PD-L1) RNA expression in PAXgene blood samples O’Rourke, Dennis Wang, Danyi Sanchez-Garcia, Jorge F. Cusano, Maria Perella Miller, Waldemar Cai, Ti Scheuenpflug, Juergen Feng, Zheng PLoS One Research Article Development of a clinically applicable liquid biopsy-based test for PD-L1 mRNA expression would be beneficial in providing complementary evidence to current immunohistochemistry assays. Hence, we report the development of a fit-for-purpose assay for detection of blood PD-L1 mRNA expression using droplet digital polymerase chain reaction (ddPCR). TaqMan® assays were selected based on coverage of the PD-L1 gene and were tested for linearity and efficiency using real-time quantitative PCR. Four reference genes were analyzed in positive control cell line (A549 treated with interferon gamma, [IFN γ]) genomic DNA. The PD-L1 primer/probe sets were also evaluated in ddPCR for limit of blank, limit of detection, and precision. Finally, thirty-five healthy volunteer samples were evaluated to establish a baseline level of PD-L1 expression. In ddPCR, the limit of blank was determined to be 0 copies and the limit of detection was determined to be less than or equal to 19 copies of PD-L1. The average intra-run coefficient of variation in the ddPCR assay was 7.44% and average inter-run CV was 7.70%. Treatment of A549 cells with IFN γ resulted in a 6.7-fold increase in PD-L1 expression (21,580 copies in untreated cDNA versus 145,000 copies in treated cDNA). Analysis of healthy human samples yielded a median value of 1659 PD-L1 copies/μL with a range of 768–7510 copies/μL. The assay was transferred to an external service provider and results from our in-house experiments and those conducted externally shows a correlation of 0.994. In conclusion, a fit-for-purpose liquid biopsy-based, purely quantitative ddPCR assay for the detection of PD-L1 mRNA expression was developed and validated using PAXgene RNA blood samples. Linearity, reproducibility, limit of blank and limit of detection were measured and deemed suitable for clinical application. This ultra-sensitive liquid biopsy ddPCR assay has promising clinical potential in screening, longitudinal monitoring and disease progression detection. Public Library of Science 2021-05-10 /pmc/articles/PMC8109819/ /pubmed/33970922 http://dx.doi.org/10.1371/journal.pone.0250849 Text en © 2021 O’Rourke et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article O’Rourke, Dennis Wang, Danyi Sanchez-Garcia, Jorge F. Cusano, Maria Perella Miller, Waldemar Cai, Ti Scheuenpflug, Juergen Feng, Zheng Fit-for-purpose quantitative liquid biopsy based droplet digital PCR assay development for detection of programmed cell death ligand-1 (PD-L1) RNA expression in PAXgene blood samples |
title | Fit-for-purpose quantitative liquid biopsy based droplet digital PCR assay development for detection of programmed cell death ligand-1 (PD-L1) RNA expression in PAXgene blood samples |
title_full | Fit-for-purpose quantitative liquid biopsy based droplet digital PCR assay development for detection of programmed cell death ligand-1 (PD-L1) RNA expression in PAXgene blood samples |
title_fullStr | Fit-for-purpose quantitative liquid biopsy based droplet digital PCR assay development for detection of programmed cell death ligand-1 (PD-L1) RNA expression in PAXgene blood samples |
title_full_unstemmed | Fit-for-purpose quantitative liquid biopsy based droplet digital PCR assay development for detection of programmed cell death ligand-1 (PD-L1) RNA expression in PAXgene blood samples |
title_short | Fit-for-purpose quantitative liquid biopsy based droplet digital PCR assay development for detection of programmed cell death ligand-1 (PD-L1) RNA expression in PAXgene blood samples |
title_sort | fit-for-purpose quantitative liquid biopsy based droplet digital pcr assay development for detection of programmed cell death ligand-1 (pd-l1) rna expression in paxgene blood samples |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8109819/ https://www.ncbi.nlm.nih.gov/pubmed/33970922 http://dx.doi.org/10.1371/journal.pone.0250849 |
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