Webinar: Analysis of Central Nervous System Tumor ctDNA from Cerebrospinal Fluid

Recent advances in molecular profiling have reshaped how central nervous system (CNS) tumors are studied and categorized. The 2021 WHO 5th Edition classification emphasizes molecular features, such as alterations in IDH1/2, TP53, BRAF, and ATRX, as essential components for tumor classification and stratification.

Conventional approaches used to evaluate CNS tumors, including MRI, cerebrospinal fluid (CSF) cytology, and tissue sampling, each present notable technical and interpretive challenges. MRI provides valuable anatomical information but has limited ability to reliably differentiate tumor subtypes. CSF cytology is constrained by low detection rates and variability in interpretation, while tissue sampling requires invasive procedures that may be difficult to perform for lesions located in sensitive or hard-to-access regions of the brain. These limitations highlight the need for alternative molecular approaches that are minimally invasive, highly informative, and suitable for repeated sampling over time.

The aim of this study was to assess the feasibility, sensitivity, and analytical value of profiling circulating tumor DNA (ctDNA) from CSF as a liquid biopsy strategy for CNS tumors. Performance was evaluated in comparison with ctDNA analysis from plasma and with matched tumor tissue sequencing. CSF, plasma, and tumor tissue samples from individuals with primary and metastatic CNS tumors were analyzed using digital PCR (dPCR) and next-generation sequencing (NGS). NGS genomic profiling was performed using Thermo Fisher’s Oncomine Precision Assay on the Genexus platform, while dPCR assays targeted well-characterized variants, including IDH1 R132H, BRAF V600E, and EGFR exon 19 deletions. The study focused on assessing concordance between tissue and liquid biopsy profiles and on determining whether longitudinal CSF ctDNA measurements could capture molecular changes over time.

Plasma-based ctDNA analysis showed limited utility for CNS tumors, consistent with the restricted transfer of tumor-derived DNA across the blood–brain barrier. In contrast, ctDNA isolated from CSF demonstrated strong agreement with the mutational landscapes observed in matched tumor tissues from gliomas, as well as lung and breast cancer metastases to the CNS. Analysis of the CSF supernatant consistently outperformed the cellular fraction, as DNA from non-tumor immune cells in the pellet reduced sensitivity.

Frequently observed alterations included IDH1 R132H, TP53, BRAF V600E, EGFR T790M, PIK3CA, and ERBB2 (HER2). Longitudinal sampling revealed shifts in ctDNA variant allele frequencies over time, suggesting that CSF ctDNA levels may reflect evolving tumor dynamics or responses to intervention. High analytical specificity was observed, with no false-positive calls in control samples, and optimal performance was achieved with input DNA quantities of at least 20 ng.

Overall, this study demonstrates that CSF-derived ctDNA is a robust and informative source for identifying tumor-associated genetic alterations in CNS tumors. The approach shows strong concordance with tissue-based sequencing and clearly outperforms plasma-based liquid biopsy methods in this setting. Importantly, CSF ctDNA analysis enables real-time molecular tracking of tumor evolution. From a translational standpoint, this strategy may reduce reliance on invasive tissue sampling and provide a practical framework for longitudinal molecular studies of CNS tumors, particularly in cases where traditional sampling is limited by anatomical or procedural constraints.