Liquid Biopsy of Brain Tumors: Spinal Fluid DNA Profiling Moves Into Real-World Neuro-Oncology

Brain tumors occupy one of the most diagnostically awkward positions in all of medicine. They sit behind the blood-brain barrier, sheltered from the bloodstream, and are typically reachable only through neurosurgery. When a suspicious lesion appears on an MRI scan, clinicians must often decide whether to subject a patient to a needle biopsy — a procedure that carries risks of hemorrhage, stroke, and injury to eloquent brain regions, and which fails to yield a diagnosis in roughly 10 to 15 percent of cases. A new study published in the Journal of Neuro-Oncology now offers one of the most detailed real-world assessments yet of an alternative: sequencing fragments of tumor DNA that float freely in cerebrospinal fluid, the clear liquid that bathes the brain and spinal cord.

The research, conducted at Mayo Clinic in Rochester, Minnesota, describes the institution’s early experience deploying a research-based cerebrospinal fluid (CSF) cell-free DNA profiling program across its neurology, oncology, and neurosurgery departments. Beginning in August 2021 and continuing through December 2024, any clinician performing a clinically indicated lumbar puncture could request that the fluid be analyzed for tumor-derived genetic material. Seventy-six consecutive patients were enrolled under an institutional review board-approved protocol, with informed consent obtained for the brain tumor biomarkers study and for storage in the Mayo Clinic Neuro-Oncology biorepository. The result is a portrait of liquid biopsy testing as it actually happens in a busy hospital — messy, heterogeneous, and full of both promise and practical constraints.

The technical pipeline behind the study is worth understanding, because it illustrates the engineering challenge of detecting vanishingly small amounts of tumor DNA. After collection, CSF samples were kept on ice and centrifuged within one to two hours, then frozen and shipped to Predicine, Inc., where cell-free DNA was extracted using a silica-membrane kit and quantified with a fluorometer and fragment analyzer. The median yield was just 0.27 nanograms per milliliter — a tiny quantity compared with what is typically recovered from blood plasma. Depending on the amount and quality of DNA recovered, samples then underwent either targeted next-generation sequencing of a 152-gene cancer panel on an Illumina NovaSeq 6000, low-pass whole-genome sequencing to generate genome-wide copy-number profiles, or both. Error suppression was achieved by merging sequencing reads from the same DNA molecules into consensus sequences, and a variant was only called as positive if it was supported by at least three independent DNA fragments, including at least one duplex fragment, and met a stringent log-odds threshold.

Patients fell into three broad clinical categories, each representing a classic diagnostic dilemma in neuro-oncology. Forty-six percent had a brain lesion of unknown origin seen on MRI that had not yet been biopsied. Twenty-one percent had a known glioma or other tumor, and their clinicians were trying to distinguish true tumor progression from treatment-related changes such as radiation necrosis or pseudoprogression — a distinction that conventional MRI handles poorly, with sensitivity and specificity ranging from only 30 to 70 percent. The remaining 26 percent had known systemic cancer with new neurological findings, and the question was whether leptomeningeal disease — the spread of cancer into the membranes lining the brain and spinal cord — was present. Cytology, the traditional test for that condition, is notoriously insensitive: in this cohort it was negative in 16 of the 18 patients in whom it was obtained.

Across all three indications, the assay produced what the researchers call a positive tumor call — detection of a cancer-associated variant allele or copy-number alteration — in 20 of the 76 patients, or 26 percent. Another 29 patients, 38 percent, had adequate DNA but no detectable tumor signal, while 27 patients, 36 percent, had insufficient cfDNA yield or quality to permit analysis. Notably, the distribution of positive, negative, and uninformative results was remarkably similar across the three clinical questions, suggesting that the assay behaves consistently regardless of why it is ordered. Only two of the sequencing runs failed quality control, a low failure rate that the authors attribute to careful sample handling and the robustness of the extraction workflow.

One of the study’s most striking findings concerns what predicts success. Increasing the volume of CSF collected did not correlate with the amount of DNA recovered — a counterintuitive result that underscores how heterogeneous tumor DNA shedding is among patients. Instead, positive tumor calls were strongly associated with higher cfDNA concentration itself: samples with detectable tumor signals had a median yield of 0.91 nanograms per milliliter, compared with 0.30 for negative samples and essentially zero for the low-yield group. Tumor proximity to the CSF space is thought to be a major driver of this variability, since fully embedded parenchymal lesions with little contact with CSF are unlikely to release detectable DNA into the fluid. This means that a negative result must always be interpreted in light of the lesion’s anatomy, not as definitive proof of absence of disease.

When tumor DNA was detected, it told clinically meaningful stories. Low-pass whole-genome sequencing uncovered the 1p/19q co-deletion characteristic of oligodendroglioma in one patient and the chromosome 7 gain and chromosome 10 loss typical of glioblastoma in others. Targeted sequencing revealed mutations in genes including TP53, TERT, IDH1, BRAF, EGFR, and MYD88 — the latter a disease-defining alteration in primary central nervous system lymphoma. In the three patients who had also undergone clinical tissue sequencing, CSF and tissue shared a substantial fraction of mutations, but each patient also carried mutations found only in the spinal fluid, a phenomenon the authors attribute to spatial heterogeneity within tumors or to genomic evolution between the times of tissue and fluid sampling.

The study also identified a quantitative biomarker with prognostic power. By summing genome-wide copy-number deviations into a single copy-number burden score, the team found that a threshold of 7.42 cleanly separated samples with true tumor-specific copy-number alterations from those without, achieving an area under the receiver operating curve of 0.998. Patients whose CSF exceeded that threshold had significantly worse overall survival, with a hazard ratio of 3.4 after controlling for the clinical indication. More broadly, patients with any positive tumor call survived significantly less long than those with negative calls or low DNA yield — a difference driven largely by the unknown-diagnosis group, with a similar trend among patients being evaluated for leptomeningeal disease. In other words, even a negative result carries information: abundant tumor DNA in spinal fluid is a bad sign, while its absence, in the right clinical context, is reassuring.

Three case vignettes bring the statistics to life. A 72-year-old man with chronic lymphocytic leukemia presented with confusion and diffuse brain abnormalities on MRI; his CSF revealed chromosome 12 amplification consistent with trisomy 12 CLL, plus KRAS and BRAF mutations, helping confirm rare central nervous system involvement of his leukemia. A 38-year-old man with metastatic gastroesophageal adenocarcinoma showed an extensive copy-number burden and TP53, NTRK1, and EGFR mutations in his spinal fluid, confirming leptomeningeal spread and guiding treatment decisions. And a 51-year-old woman with a surveillance question about her grade 3 oligodendroglioma had her tumor’s molecular signature — the 1p/19q co-deletion and IDH1 R132H mutation — detected in CSF months before imaging confirmed ventricular progression, ultimately informing her enrollment in an IDH inhibitor trial.

The authors are candid about the limitations. The copy-number burden threshold was derived post hoc without an independent validation cohort, tissue sequencing was available for only a handful of patients, and cytology’s poor sensitivity made it an imperfect gold standard for leptomeningeal disease. For progression-versus-pseudoprogression questions, the mixture of recurrent tumor and treatment effects likely explains why survival did not differ by tumor-call status in that subgroup. Still, the study demonstrates that CSF cfDNA sequencing can be deployed across the full breadth of a real neuro-oncology practice with acceptable failure rates, and that requests for testing have grown substantially since the program began. As low-input sequencing platforms mature, the authors argue, spinal fluid liquid biopsy is poised to become a routine complement — and in some cases a safer alternative — to the neurosurgeon’s needle.

Subject of Research: Cerebrospinal fluid cell-free DNA sequencing as a liquid biopsy for diagnosing and monitoring brain tumors and leptomeningeal disease in neuro-oncology practice

Article Title: Early experience with cerebrospinal fluid cell-free DNA molecular profiling in a neuro-oncology practice

Article References: Riviere-Cazaux, C., Kumar, R., Rechberger, J. S., Obiri-Yeboah, D., Dai, C., Li, J., Huang, Y., Warrington, A. E., Sharif, E. H., Palmer, E. A., Wang, X., Lachance, D. H., Kizilbash, S. H., Ruff, M. W., Carabenciov, I. D., Fortin Ensign, S. P., Zadeh, G., Neth, B. J., Sener, U., … Burns, T. C. (2026). Early experience with cerebrospinal fluid cell-free DNA molecular profiling in a neuro-oncology practice. Journal of Neuro-Oncology, 180(1), Article 2. https://doi.org/10.1007/s11060-026-05799-7

Image Credits: AI Generated

DOI: 10.1007/s11060-026-05799-7

Keywords: cerebrospinal fluid, cell-free DNA, liquid biopsy, neuro-oncology, brain tumors, glioma, leptomeningeal disease, next-generation sequencing, copy-number alterations, pseudoprogression, Mayo Clinic, biomarkers