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Methodological Challenges of Digital PCR Detection of the Histone H3 K27M Somatic Variant in Cerebrospinal Fluid

Cell-free DNA (cfDNA) in body fluids is invaluable for cancer diagnostics. Despite the impressive potential of liquid biopsies for the diagnostics of central nervous system (CNS) tumors, a number of challenges prevent introducing this approach into routine laboratory practice. In this study, we adop...

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Autores principales: Zaytseva, Margarita, Usman, Natalia, Salnikova, Ekaterina, Sanakoeva, Agunda, Valiakhmetova, Andge, Chervova, Almira, Papusha, Ludmila, Novichkova, Galina, Druy, Alexander
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9039021/
https://www.ncbi.nlm.nih.gov/pubmed/35498161
http://dx.doi.org/10.3389/pore.2022.1610024
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author Zaytseva, Margarita
Usman, Natalia
Salnikova, Ekaterina
Sanakoeva, Agunda
Valiakhmetova, Andge
Chervova, Almira
Papusha, Ludmila
Novichkova, Galina
Druy, Alexander
author_facet Zaytseva, Margarita
Usman, Natalia
Salnikova, Ekaterina
Sanakoeva, Agunda
Valiakhmetova, Andge
Chervova, Almira
Papusha, Ludmila
Novichkova, Galina
Druy, Alexander
author_sort Zaytseva, Margarita
collection PubMed
description Cell-free DNA (cfDNA) in body fluids is invaluable for cancer diagnostics. Despite the impressive potential of liquid biopsies for the diagnostics of central nervous system (CNS) tumors, a number of challenges prevent introducing this approach into routine laboratory practice. In this study, we adopt a protocol for sensitive detection of the H3 K27M somatic variant in cerebrospinal fluid (CSF) by using digital polymerase chain reaction (dPCR). Optimization of the protocol was carried out stepwise, including preamplification of the H3 target region and adjustment of dPCR conditions. The optimized protocol allowed detection of the mutant allele starting from DNA quantities as low as 9 picograms. Analytical specificity was tested using a representative group of tumor tissue samples with known H3 K27M status, and no false-positive cases were detected. The protocol was applied to a series of CSF samples collected from patients with CNS tumors (n = 18) using two alternative dPCR platforms, QX200 Droplet Digital PCR system (Bio-Rad) and QIAcuity Digital PCR System (Qiagen). In three out of four CSF specimens collected from patients with H3 K27M-positive diffuse midline glioma, both platforms allowed detection of the mutant allele. The use of ventricular access for CSF collection appears preferential, as lumbar CSF samples may produce ambiguous results. All CSF samples collected from patients with H3 wild-type tumors were qualified as H3 K27M-negative. High agreement of the quantitative data obtained with the two platforms demonstrates universality of the approach.
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spelling pubmed-90390212022-04-27 Methodological Challenges of Digital PCR Detection of the Histone H3 K27M Somatic Variant in Cerebrospinal Fluid Zaytseva, Margarita Usman, Natalia Salnikova, Ekaterina Sanakoeva, Agunda Valiakhmetova, Andge Chervova, Almira Papusha, Ludmila Novichkova, Galina Druy, Alexander Pathol Oncol Res Pathology and Oncology Archive Cell-free DNA (cfDNA) in body fluids is invaluable for cancer diagnostics. Despite the impressive potential of liquid biopsies for the diagnostics of central nervous system (CNS) tumors, a number of challenges prevent introducing this approach into routine laboratory practice. In this study, we adopt a protocol for sensitive detection of the H3 K27M somatic variant in cerebrospinal fluid (CSF) by using digital polymerase chain reaction (dPCR). Optimization of the protocol was carried out stepwise, including preamplification of the H3 target region and adjustment of dPCR conditions. The optimized protocol allowed detection of the mutant allele starting from DNA quantities as low as 9 picograms. Analytical specificity was tested using a representative group of tumor tissue samples with known H3 K27M status, and no false-positive cases were detected. The protocol was applied to a series of CSF samples collected from patients with CNS tumors (n = 18) using two alternative dPCR platforms, QX200 Droplet Digital PCR system (Bio-Rad) and QIAcuity Digital PCR System (Qiagen). In three out of four CSF specimens collected from patients with H3 K27M-positive diffuse midline glioma, both platforms allowed detection of the mutant allele. The use of ventricular access for CSF collection appears preferential, as lumbar CSF samples may produce ambiguous results. All CSF samples collected from patients with H3 wild-type tumors were qualified as H3 K27M-negative. High agreement of the quantitative data obtained with the two platforms demonstrates universality of the approach. Frontiers Media S.A. 2022-04-12 /pmc/articles/PMC9039021/ /pubmed/35498161 http://dx.doi.org/10.3389/pore.2022.1610024 Text en Copyright © 2022 Zaytseva, Usman, Salnikova, Sanakoeva, Valiakhmetova, Chervova, Papusha, Novichkova and Druy. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Pathology and Oncology Archive
Zaytseva, Margarita
Usman, Natalia
Salnikova, Ekaterina
Sanakoeva, Agunda
Valiakhmetova, Andge
Chervova, Almira
Papusha, Ludmila
Novichkova, Galina
Druy, Alexander
Methodological Challenges of Digital PCR Detection of the Histone H3 K27M Somatic Variant in Cerebrospinal Fluid
title Methodological Challenges of Digital PCR Detection of the Histone H3 K27M Somatic Variant in Cerebrospinal Fluid
title_full Methodological Challenges of Digital PCR Detection of the Histone H3 K27M Somatic Variant in Cerebrospinal Fluid
title_fullStr Methodological Challenges of Digital PCR Detection of the Histone H3 K27M Somatic Variant in Cerebrospinal Fluid
title_full_unstemmed Methodological Challenges of Digital PCR Detection of the Histone H3 K27M Somatic Variant in Cerebrospinal Fluid
title_short Methodological Challenges of Digital PCR Detection of the Histone H3 K27M Somatic Variant in Cerebrospinal Fluid
title_sort methodological challenges of digital pcr detection of the histone h3 k27m somatic variant in cerebrospinal fluid
topic Pathology and Oncology Archive
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9039021/
https://www.ncbi.nlm.nih.gov/pubmed/35498161
http://dx.doi.org/10.3389/pore.2022.1610024
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