Standardization of the liquid biopsy for pediatric diffuse midline glioma using ddPCR

Abstract Diffuse midline glioma (DMG) is a highly morbid pediatric brain tumor. Up to 80% of DMGs harbor mutations in histone H3-encoding genes, associated with poor prognosis. We previously showed the feasibility of detecting H3 mutations in circulating tumor DNA (ctDNA) in the liquid biome of chil...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Daphne Li, Erin R. Bonner, Kyle Wierzbicki, Eshini Panditharatna, Tina Huang, Rishi Lulla, Sabine Mueller, Carl Koschmann, Javad Nazarian, Amanda M. Saratsis
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/ae3730eeab0b412cb0317c84f0217019
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:ae3730eeab0b412cb0317c84f0217019
record_format	dspace
spelling	oai:doaj.org-article:ae3730eeab0b412cb0317c84f02170192021-12-02T13:30:10ZStandardization of the liquid biopsy for pediatric diffuse midline glioma using ddPCR10.1038/s41598-021-84513-12045-2322https://doaj.org/article/ae3730eeab0b412cb0317c84f02170192021-03-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-84513-1https://doaj.org/toc/2045-2322Abstract Diffuse midline glioma (DMG) is a highly morbid pediatric brain tumor. Up to 80% of DMGs harbor mutations in histone H3-encoding genes, associated with poor prognosis. We previously showed the feasibility of detecting H3 mutations in circulating tumor DNA (ctDNA) in the liquid biome of children diagnosed with DMG. However, detection of low levels of ctDNA is highly dependent on platform sensitivity and sample type. To address this, we optimized ctDNA detection sensitivity and specificity across two commonly used digital droplet PCR (ddPCR) platforms (RainDance and BioRad), and validated methods for detecting H3F3A c.83A > T (H3.3K27M) mutations in DMG CSF, plasma, and primary tumor specimens across three different institutions. DNA was extracted from H3.3K27M mutant and H3 wildtype (H3WT) specimens, including H3.3K27M tumor tissue (n = 4), CSF (n = 6), plasma (n = 4), and human primary pediatric glioma cells (H3.3K27M, n = 2; H3WT, n = 1). ctDNA detection was enhanced via PCR pre-amplification and use of distinct custom primers and fluorescent LNA probes for c.83 A > T H3F3A mutation detection. Mutation allelic frequency (MAF) was determined and validated through parallel analysis of matched H3.3K27M tissue specimens (n = 3). We determined technical nuances between ddPCR instruments, and optimized sample preparation and sequencing protocols for H3.3K27M mutation detection and quantification. We observed 100% sensitivity and specificity for mutation detection in matched DMG tissue and CSF across assays, platforms and institutions. ctDNA is reliably and reproducibly detected in the liquid biome using ddPCR, representing a clinically feasible, reproducible, and minimally invasive approach for DMG diagnosis, molecular subtyping and therapeutic monitoring.Daphne LiErin R. BonnerKyle WierzbickiEshini PanditharatnaTina HuangRishi LullaSabine MuellerCarl KoschmannJavad NazarianAmanda M. SaratsisNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Daphne Li Erin R. Bonner Kyle Wierzbicki Eshini Panditharatna Tina Huang Rishi Lulla Sabine Mueller Carl Koschmann Javad Nazarian Amanda M. Saratsis Standardization of the liquid biopsy for pediatric diffuse midline glioma using ddPCR
description	Abstract Diffuse midline glioma (DMG) is a highly morbid pediatric brain tumor. Up to 80% of DMGs harbor mutations in histone H3-encoding genes, associated with poor prognosis. We previously showed the feasibility of detecting H3 mutations in circulating tumor DNA (ctDNA) in the liquid biome of children diagnosed with DMG. However, detection of low levels of ctDNA is highly dependent on platform sensitivity and sample type. To address this, we optimized ctDNA detection sensitivity and specificity across two commonly used digital droplet PCR (ddPCR) platforms (RainDance and BioRad), and validated methods for detecting H3F3A c.83A > T (H3.3K27M) mutations in DMG CSF, plasma, and primary tumor specimens across three different institutions. DNA was extracted from H3.3K27M mutant and H3 wildtype (H3WT) specimens, including H3.3K27M tumor tissue (n = 4), CSF (n = 6), plasma (n = 4), and human primary pediatric glioma cells (H3.3K27M, n = 2; H3WT, n = 1). ctDNA detection was enhanced via PCR pre-amplification and use of distinct custom primers and fluorescent LNA probes for c.83 A > T H3F3A mutation detection. Mutation allelic frequency (MAF) was determined and validated through parallel analysis of matched H3.3K27M tissue specimens (n = 3). We determined technical nuances between ddPCR instruments, and optimized sample preparation and sequencing protocols for H3.3K27M mutation detection and quantification. We observed 100% sensitivity and specificity for mutation detection in matched DMG tissue and CSF across assays, platforms and institutions. ctDNA is reliably and reproducibly detected in the liquid biome using ddPCR, representing a clinically feasible, reproducible, and minimally invasive approach for DMG diagnosis, molecular subtyping and therapeutic monitoring.
format	article
author	Daphne Li Erin R. Bonner Kyle Wierzbicki Eshini Panditharatna Tina Huang Rishi Lulla Sabine Mueller Carl Koschmann Javad Nazarian Amanda M. Saratsis
author_facet	Daphne Li Erin R. Bonner Kyle Wierzbicki Eshini Panditharatna Tina Huang Rishi Lulla Sabine Mueller Carl Koschmann Javad Nazarian Amanda M. Saratsis
author_sort	Daphne Li
title	Standardization of the liquid biopsy for pediatric diffuse midline glioma using ddPCR
title_short	Standardization of the liquid biopsy for pediatric diffuse midline glioma using ddPCR
title_full	Standardization of the liquid biopsy for pediatric diffuse midline glioma using ddPCR
title_fullStr	Standardization of the liquid biopsy for pediatric diffuse midline glioma using ddPCR
title_full_unstemmed	Standardization of the liquid biopsy for pediatric diffuse midline glioma using ddPCR
title_sort	standardization of the liquid biopsy for pediatric diffuse midline glioma using ddpcr
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/ae3730eeab0b412cb0317c84f0217019
work_keys_str_mv	AT daphneli standardizationoftheliquidbiopsyforpediatricdiffusemidlinegliomausingddpcr AT erinrbonner standardizationoftheliquidbiopsyforpediatricdiffusemidlinegliomausingddpcr AT kylewierzbicki standardizationoftheliquidbiopsyforpediatricdiffusemidlinegliomausingddpcr AT eshinipanditharatna standardizationoftheliquidbiopsyforpediatricdiffusemidlinegliomausingddpcr AT tinahuang standardizationoftheliquidbiopsyforpediatricdiffusemidlinegliomausingddpcr AT rishilulla standardizationoftheliquidbiopsyforpediatricdiffusemidlinegliomausingddpcr AT sabinemueller standardizationoftheliquidbiopsyforpediatricdiffusemidlinegliomausingddpcr AT carlkoschmann standardizationoftheliquidbiopsyforpediatricdiffusemidlinegliomausingddpcr AT javadnazarian standardizationoftheliquidbiopsyforpediatricdiffusemidlinegliomausingddpcr AT amandamsaratsis standardizationoftheliquidbiopsyforpediatricdiffusemidlinegliomausingddpcr
_version_	1718392995053568000

Standardization of the liquid biopsy for pediatric diffuse midline glioma using ddPCR

Ejemplares similares