Abstract
PurposeConventional genetic analyzers require surgically obtained tumor tissues to confirm the molecular diagnosis of diffuse glioma. Recent technical breakthroughs have enabled increased utilization of cell-free tumor DNA (ctDNA) in body fluids as a reliable resource for molecular diagnosis in various cancers. Here, we tested the application of a chip-based digital PCR system for the less invasive diagnosis (i.e., liquid biopsy) of diffuse glioma using the cerebrospinal fluid (CSF).MethodsCSF samples from 34 patients with diffuse glioma were collected from the surgical field during craniotomy. Preoperative lumbar CSF collection was also performed in 11 patients. Extracted ctDNA was used to analyze diagnostic point mutations in IDH1 R132H, TERT promoter (C228T and C250T), and H3F3A (K27M) on the QuantStudio® 3D Digital PCR System. These results were compared with their corresponding tumor DNA samples.ResultsWe detected either of the diagnostic mutations in tumor DNA samples from 28 of 34 patients. Among them, we achieved precise molecular diagnoses using intracranial CSF in 20 (71%). Univariate analyses revealed that the World Health Organization (WHO) grade (p = 0.0034), radiographic enhancement (p = 0.0006), and Mib1 index (p = 0.01) were significant predictors of precise CSF-based molecular diagnosis. We precisely diagnosed WHO grade III or IV diffuse gliomas using lumbar CSF obtained from 6 (87%) of 7 patients with tumors harboring any mutation.ConclusionWe established a novel, non-invasive molecular diagnostic method using a chip-based digital PCR system targeting ctDNA derived from CSF with high sensitivity and specificity, especially for high-grade gliomas.
Highlights
Molecular diagnosis of diffuse gliomas is conventionally performed using surgically obtained tumor tissues; the 2016 central nervous system (CNS) World Health Organization (WHO) classification defined a novel tumor [diffuse midline glioma, H3F3A K27M (H3 K27M) mutation (DMG, H3 K27M-mut)], diagnosis of which was confirmed by molecular findings [1]
Recent reports revealed the presence of cell-free tumor DNA in the cerebrospinal fluid (CSF) obtained from patients with CNS tumors [6,7,8], and ctDNA is used as a target for liquid biopsy of diffuse glioma [9,10,11]
To verify the reproducibility of PCR products amplified from CSF-derived cell-free DNA (cfDNA), two pairs of cfDNA and the corresponding tumor-tissue DNA obtained from different patients were subjected to gel electrophoresis (Fig. 1)
Summary
Molecular diagnosis of diffuse gliomas is conventionally performed using surgically obtained tumor tissues; the 2016 central nervous system (CNS) World Health Organization (WHO) classification defined a novel tumor [diffuse midline glioma, H3 K27M mutation (DMG, H3 K27M-mut)], diagnosis of which was confirmed by molecular findings [1]. Recent reports revealed the presence of cell-free tumor (ct) DNA in the cerebrospinal fluid (CSF) obtained from patients with CNS tumors [6,7,8], and ctDNA is used as a target for liquid biopsy of diffuse glioma [9,10,11]. This promising approach promotes the utilization of molecular findings as diagnostic markers of diffuse gliomas for monitoring disease state and the development of non-operative diagnostic procedures. We evaluated the feasibility of this assay using CSF samples from patients with diffuse glioma
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