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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Published in | Pathology oncology research Vol. 28; p. 1610024 |
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Main Authors | , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
Switzerland
Frontiers Media S.A
12.04.2022
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Subjects | |
Online Access | Get full text |
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Summary: | 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
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
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
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 (
= 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
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
wild-type tumors were qualified as
K27M-negative. High agreement of the quantitative data obtained with the two platforms demonstrates universality of the approach. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Edited by: József Tímár, Semmelweis University, Hungary |
ISSN: | 1532-2807 1219-4956 1532-2807 |
DOI: | 10.3389/pore.2022.1610024 |