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...

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Autores principales:	Dennis O'Rourke, Danyi Wang, Jorge F Sanchez-Garcia, Maria Perella Cusano, Waldemar Miller, Ti Cai, Juergen Scheuenpflug, Zheng Feng
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5cb6fb0ac0a54dee8a6a076bc6158c73
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spelling	oai:doaj.org-article:5cb6fb0ac0a54dee8a6a076bc6158c732021-12-02T20:11:25ZFit-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.1932-620310.1371/journal.pone.0250849https://doaj.org/article/5cb6fb0ac0a54dee8a6a076bc6158c732021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0250849https://doaj.org/toc/1932-6203Development 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.Dennis O'RourkeDanyi WangJorge F Sanchez-GarciaMaria Perella CusanoWaldemar MillerTi CaiJuergen ScheuenpflugZheng FengPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 5, p e0250849 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Dennis O'Rourke Danyi Wang Jorge F Sanchez-Garcia Maria Perella Cusano Waldemar Miller Ti Cai Juergen Scheuenpflug Zheng Feng 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.
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	article
author	Dennis O'Rourke Danyi Wang Jorge F Sanchez-Garcia Maria Perella Cusano Waldemar Miller Ti Cai Juergen Scheuenpflug Zheng Feng
author_facet	Dennis O'Rourke Danyi Wang Jorge F Sanchez-Garcia Maria Perella Cusano Waldemar Miller Ti Cai Juergen Scheuenpflug Zheng Feng
author_sort	Dennis O'Rourke
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_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_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_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.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/5cb6fb0ac0a54dee8a6a076bc6158c73
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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.

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