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...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-04-01
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| Series: | Pathology and Oncology Research |
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| Online Access: | https://www.por-journal.com/articles/10.3389/pore.2022.1610024/full |
| _version_ | 1797218150732791808 |
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| author | Margarita Zaytseva Natalia Usman Ekaterina Salnikova Agunda Sanakoeva Andge Valiakhmetova Almira Chervova Almira Chervova Ludmila Papusha Galina Novichkova Alexander Druy Alexander Druy |
| author_facet | Margarita Zaytseva Natalia Usman Ekaterina Salnikova Agunda Sanakoeva Andge Valiakhmetova Almira Chervova Almira Chervova Ludmila Papusha Galina Novichkova Alexander Druy Alexander Druy |
| author_sort | Margarita Zaytseva |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-04-24T12:13:11Z |
| format | Article |
| id | doaj.art-0e0969f8cf7043998061dfa8202ee8b6 |
| institution | Directory Open Access Journal |
| issn | 1532-2807 |
| language | English |
| last_indexed | 2024-04-24T12:13:11Z |
| publishDate | 2022-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Pathology and Oncology Research |
| spelling | doaj.art-0e0969f8cf7043998061dfa8202ee8b62024-04-08T09:32:19ZengFrontiers Media S.A.Pathology and Oncology Research1532-28072022-04-012810.3389/pore.2022.16100241610024Methodological Challenges of Digital PCR Detection of the Histone H3 K27M Somatic Variant in Cerebrospinal FluidMargarita Zaytseva0Natalia Usman1Ekaterina Salnikova2Agunda Sanakoeva3Andge Valiakhmetova4Almira Chervova5Almira Chervova6Ludmila Papusha7Galina Novichkova8Alexander Druy9Alexander Druy10Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, RussiaDmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, RussiaDmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, RussiaDmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, RussiaDmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, RussiaDmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, RussiaEpigenomics, Proliferation, and the Identity of Cells, Department of Developmental and Stem Cell Biology, Institut Pasteur, Paris, FranceDmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, RussiaDmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, RussiaDmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, RussiaResearch Institute of Medical Cell Technologies, Yekaterinburg, RussiaCell-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.https://www.por-journal.com/articles/10.3389/pore.2022.1610024/fullcerebrospinal fluidliquid biopsydPCRcell-free DNACNS tumordiffuse midline glioma |
| spellingShingle | Margarita Zaytseva Natalia Usman Ekaterina Salnikova Agunda Sanakoeva Andge Valiakhmetova Almira Chervova Almira Chervova Ludmila Papusha Galina Novichkova Alexander Druy Alexander Druy Methodological Challenges of Digital PCR Detection of the Histone H3 K27M Somatic Variant in Cerebrospinal Fluid Pathology and Oncology Research cerebrospinal fluid liquid biopsy dPCR cell-free DNA CNS tumor diffuse midline glioma |
| 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 | cerebrospinal fluid liquid biopsy dPCR cell-free DNA CNS tumor diffuse midline glioma |
| url | https://www.por-journal.com/articles/10.3389/pore.2022.1610024/full |
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