Abstract
Diffuse midline gliomas (including diffuse intrinsic pontine glioma, DIPG) are highly morbid glial neoplasms of the thalamus or brainstem that typically arise in young children and are not surgically resectable. These tumors are characterized by a high rate of histone H3 mutation, resulting in replacement of lysine 27 with methionine (K27M) in genes encoding H3 variants H3.3 (H3F3A) and H3.1 (HIST1H3B). Detection of these gain-of-function mutations has clinical utility, as they are associated with distinct tumor biology and clinical outcomes. Given the paucity of tumor tissue available for molecular analysis and relative morbidity of midline tumor biopsy, CSF-derived tumor DNA from patients with diffuse midline glioma may serve as a viable alternative for clinical detection of histone H3 mutation. We demonstrate the feasibility of two strategies to detect H3 mutations in CSF-derived tumor DNA from children with brain tumors (n = 11) via either targeted Sanger sequencing of H3F3A and HIST1H3B, or H3F3A c.83 A > T detection via nested PCR with mutation-specific primers. Of the six CSF specimens from children with diffuse midline glioma in our cohort, tumor DNA sufficient in quantity and quality for analysis was isolated from five (83%), with H3.3K27M detected in four (66.7%). In addition, H3.3G34V was identified in tumor DNA from a patient with supratentorial glioblastoma. Test sensitivity (87.5%) and specificity (100%) was validated via immunohistochemical staining and Sanger sequencing in available matched tumor tissue specimens (n = 8). Our results indicate that histone H3 gene mutation is detectable in CSF-derived tumor DNA from children with brain tumors, including diffuse midline glioma, and suggest the feasibility of “liquid biopsy” in lieu of, or to complement, tissue diagnosis, which may prove valuable for stratification to targeted therapies and monitoring treatment response.
Highlights
Diffuse midline gliomas are high-grade glial neoplasms of the thalamus or brainstem, and occur almost exclusively in young children
Comparison of nucleic acid precipitation carriers To compare the efficacy of carrier RNA and linear polyacrylamide (LPA) as nucleic acid precipitation carriers, we used each carrier to extract DNA from matched cerebrospinal fluid (CSF) aliquots from four patients (Table 1)
We observed no significant difference (p = 0.97) in Sanger sequencing results or the amount of nucleic acid extracted using carrier RNA compared to LPA. These data demonstrate that LPA does not compromise the yield or quality of the nucleic acid isolated from CSF
Summary
Diffuse midline gliomas are high-grade glial neoplasms of the thalamus or brainstem (including diffuse intrinsic pontine glioma, DIPG), and occur almost exclusively in young children. Given the biological and clinical implications of histone H3 mutation in diffuse midline glioma, mutation detection is of great interest for advancing understanding of tumor biology and improving patient treatment Biopsy of these tumors for genetic analysis is not without clinical risk [30]. We set to detect Histone H3 mutation in archival CSF collected from pediatric patients with diffuse midline glioma, including DIPG, and to validate these findings in patient-derived tumor tissue This approach could serve as a safe and robust method of “liquid biopsy” for histone H3 mutation detection in children with diffuse midline glioma, to potentially facilitate clinical stratification to targeted therapies and measure response to treatment
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