Next-generation sequencing of cerebrospinal fluid for clinical molecular diagnostics in pediatric, adolescent and young adult brain tumor patients

Abstract

Background: Safe sampling of central nervous system tumor tissue for diagnostic purposes may be difficult if not impossible, especially in pediatric patients, and an unmet need exists to develop less invasive diagnostic tests.

Methods: We report our clinical experience with minimally invasive molecular diagnostics using a clinically validated assay for sequencing of cerebrospinal fluid (CSF) cell-free DNA (cfDNA). All CSF samples were collected as part of clinical care, and results reported to both clinicians and patients/families.

Results: We analyzed 64 CSF samples from 45 pediatric, adolescent and young adult (AYA) patients (pediatric = 25; AYA = 20) with primary and recurrent brain tumors across 12 histopathological subtypes including high-grade glioma (n = 10), medulloblastoma (n = 10), pineoblastoma (n = 5), low-grade glioma (n = 4), diffuse leptomeningeal glioneuronal tumor (DLGNT) (n = 4), retinoblastoma (n = 4), ependymoma (n = 3), and other (n = 5). Somatic alterations were detected in 30/64 samples (46.9%) and in at least one sample per unique patient in 21/45 patients (46.6%). CSF cfDNA positivity was strongly associated with the presence of disseminated disease at the time of collection (81.5% of samples from patients with disseminated disease were positive). No association was seen between CSF cfDNA positivity and the timing of CSF collection during the patient's disease course.

Conclusions: We identified three general categories where CSF cfDNA testing provided additional relevant diagnostic, prognostic, and/or therapeutic information, impacting clinical assessment and decision making: (1) diagnosis and/or identification of actionable alterations; (2) monitor response to therapy; and (3) tracking tumor evolution. Our findings support broader implementation of clinical CSF cfDNA testing in this population to improve care.

Keywords: NextGen sequencing; cell-free DNA; cerebrospinal fluid (CSF); circulating tumor DNA; liquid biopsy; pediatric brain tumors.

Fig. 1

Frequency of CSF cfDNA detection across various pediatric CNS tumor types. Positive samples are shown in blue, negative samples in red.

Fig. 2

Oncoprint of tumor and CSF mutations across 21 pediatric patients with primary CNS tumors and positive CSF cfDNA. Shown are the most frequent genomic alterations including single nucleotide variants (SNVs), copy number alterations (CNAs), and structural variants (SVs).

Fig. 3

Concordance of mutations across tumor tissue versus CSF across matched pairs. Shown is the frequency per patient of shared versus tissue-only or CSF-only mutations in matched tumor tissue—CSF sample pairs (N = 15 patient). Blue, shared; red, tissue-only; gray, CSF-only. Concordance analysis was performed with the tumor tissue and the CSF sample obtained in closest temporal proximity to each other.