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

The research, led by Cédric Poyet of Stadtspital Triemli in Zurich and Marie Lork of the University Hospital of Zurich, together with colleagues from Kantonsspital Baden, Luzerner Kantonsspital and University Hospital Zurich, set out to identify molecular correlates of overall survival in muscle-invasive bladder cancer, often abbreviated MIBC. The team analyzed tumor DNA extracted from cystectomy specimens, the surgical samples obtained when the bladder is removed, from 32 patients treated at Swiss centers. The study received ethical approval from the Cantonal Ethics Committee Zurich and was conducted in accordance with the Declaration of Helsinki.

To characterize the genomic landscape of each tumor, the investigators used the FoundationOne CDx comprehensive genomic profiling platform, a targeted next-generation sequencing assay capable of detecting substitutions, insertions and deletions, copy number alterations and selected genomic instability markers across hundreds of cancer-related genes. Patients were then divided into two comparison groups based on a hard clinical endpoint: a favorable outcome group of 14 patients who survived at least 60 months after surgery, and a poor outcome group of 18 patients who survived fewer than 60 months. This dichotomy allowed the researchers to ask a simple but clinically vital question: which genomic features separate long-term survivors from those who die earlier of their disease?

Across the entire cohort, the sequencing effort uncovered 279 pathogenic or likely pathogenic mutations distributed across 88 genes. The most frequently altered genes were familiar names in bladder cancer biology: TP53, the guardian-of-the-genome tumor suppressor whose inactivation is a near-universal event in this disease; PIK3CA, a signaling kinase driving PI3K pathway activation; KDM6A, a histone demethylase involved in chromatin regulation; and FGFR3, a receptor tyrosine kinase that is a well-established oncogenic driver and drug target in urothelial carcinoma. Perhaps surprisingly, the distributions of these frequent alterations were similar between the favorable and poor outcome groups, indicating that the presence or absence of these canonical mutations alone does not explain the dramatic survival differences observed in the clinic.

The team next turned to the standard quantitative indicators of genomic instability that have been proposed as prognostic and predictive biomarkers in many tumor types. Tumor mutational burden, or TMB, reflects the total number of somatic mutations carried by a tumor and is widely used as a proxy for responsiveness to immune checkpoint inhibitors. Microsatellite instability, or MSI, marks defects in DNA mismatch repair and carries prognostic and predictive significance in colorectal and several other cancers. In this MIBC cohort, however, both metrics were comparable between the long-term survivors and the poor outcome group, and neither proved prognostically informative. The finding is a caution against assuming that biomarkers validated in other cancers will translate directly to bladder cancer.

The decisive signal emerged from a different layer of genomic complexity: copy number alterations. Tumors from the poor outcome group exhibited a significantly higher frequency and burden of gene amplifications, events in which segments of DNA containing particular genes are copied multiple times, often massively, driving overexpression of the encoded proteins. Crucially, these amplifications frequently involved known oncogenes and co-amplification hotspots, regions of the genome where neighboring growth-promoting genes are gained together in a single event. In other words, patients whose tumors carried a heavy load of oncogene amplifications were disproportionately represented among those who died within five years of cystectomy.

The biological logic behind this observation is compelling. While point mutations typically disable tumor suppressors or alter the function of a single protein, amplifications act as gene dosage escalators, flooding tumor cells with growth factor receptors, signaling kinases and cell cycle accelerators. High-level amplification of oncogenes can simultaneously promote proliferation, survival under therapeutic stress and metastatic competence. Moreover, co-amplification events can deliver several oncogenic payloads at once, creating tumors that are intrinsically more aggressive and harder to eradicate with a single targeted agent. The Swiss findings suggest that this dosage-driven mode of tumor evolution may be a hallmark of the most lethal forms of MIBC.

The results also carry therapeutic implications. Amplified oncogenes are, in principle, druggable targets. FGFR inhibitors are already approved for metastatic urothelial carcinoma in tumors with FGFR alterations, and agents directed against amplified receptor kinases and downstream signaling nodes are in clinical development across many cancer types. If the association between amplification burden and poor survival is confirmed, comprehensive copy number profiling at the time of cystectomy could help identify patients who warrant intensified treatment, such as perioperative systemic therapy escalation, enrollment in targeted therapy trials or closer surveillance for recurrence. Conversely, the lack of prognostic value for TMB and MSI in this cohort suggests that these markers should not be relied upon in isolation for outcome prediction in MIBC.

The authors are careful to frame the study as exploratory, and the caveats are substantial. The cohort comprised only 32 patients, divided into groups of 14 and 18, a sample size that limits statistical power and leaves open the possibility of confounding by clinical factors such as stage, nodal status and treatment sequence, which the abstract does not address in detail. The use of a targeted panel, while broad, does not capture the full spectrum of structural variants and noncoding alterations that whole-genome sequencing would reveal. The authors explicitly call for validation in larger cohorts to determine whether oncogene amplifications can serve as robust prognostic markers and to explore their potential as therapeutic targets. It is also worth noting that Roche funded the genomic testing through the FoundationOne CDx platform but had no role in study design, data analysis, interpretation or manuscript writing, apart from being granted the opportunity to review the manuscript prior to submission.

Even with these limitations, the study adds an important dimension to the ongoing effort to bring precision oncology to bladder cancer. The field has long focused on the mutational catalog of urothelial carcinoma, one of the most heavily mutated of all common tumors, yet this work suggests that the architecture of copy number gains may carry at least as much prognostic weight as the mutation list itself. For patients facing cystectomy, a procedure with significant morbidity and a five-year survival that remains unsatisfactory for many, any molecular signal that reliably separates indolent from lethal disease is valuable. If larger studies confirm that oncogene amplification burden predicts poor overall survival, clinicians may one day sequence not just for mutations but for the sheer number of oncogene copies a tumor carries, using that information to triage patients toward more aggressive, and hopefully more effective, treatment strategies from the moment of diagnosis.

Subject of Research: Genomic profiling of oncogene amplifications as prognostic markers of overall survival in muscle-invasive bladder cancer

Article Title: Oncogene-driven genomic profiles are linked to poor overall survival in muscle-invasive bladder cancer (MIBC)

Article References: Poyet, C., Franzen, A. S., Bieri, U., Kaufmann, E., Eberli, D., Schmid, M., Zoche, M., Moch, H., & Lork, M. (2026). Oncogene-driven genomic profiles are linked to poor overall survival in muscle-invasive bladder cancer (MIBC). Journal of Cancer Research and Clinical Oncology. https://doi.org/10.1007/s00432-026-06626-2

Image Credits: AI Generated

DOI: 10.1007/s00432-026-06626-2

Keywords: muscle-invasive bladder cancer, oncogene amplifications, genomic profiling, tumor mutational burden, microsatellite instability, TP53, FGFR3, PIK3CA, copy number alterations, prognostic biomarkers, cystectomy, FoundationOne CDx