Related Experiment Video
Updated: Jul 18, 2025
Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting
Published on: September 25, 2012
Classification of Brain Tumors by Nanopore Sequencing of Cell-Free DNA from Cerebrospinal Fluid
Ann-Kristin Afflerbach1,2, Christian Rohrandt3, Björn Brändl4
1Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
Background:
Molecular brain tumor diagnosis is usually dependent on tissue biopsies or resections. This can pose several risks associated with anesthesia or neurosurgery, especially for lesions in the brain stem or other difficult-to-reach anatomical sites. Apart from initial diagnosis, tumor progression, recurrence, or the acquisition of novel genetic alterations can only be proven by re-biopsies.
Methods:
We employed Nanopore sequencing on cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) and analyzed copy number variations (CNV) and global DNA methylation using a random forest classifier. We sequenced 129 samples with sufficient DNA. These samples came from 99 patients and encompassed 22 entities. Results were compared to clinical diagnosis and molecular analysis of tumor tissue, if available.
Results:
110/129 samples were technically successful, and 50 of these contained detectable circulating tumor DNA (ctDNA) by CNV or methylation profiling. ctDNA was detected in samples from patients with progressive disease but also from patients without known residual disease. CNV plots showed diagnostic and prognostic alterations, such as C19MC amplifications in embryonal tumors with multilayered rosettes or Chr.1q gains and Chr.6q losses in posterior fossa group A ependymoma, respectively. Most CNV profiles mirrored the profiles of the respective tumor tissue. DNA methylation allowed exact classification of the tumor in 22/110 cases and led to incorrect classification in 2/110 cases. Only 5/50 samples with detected ctDNA contained tumor cells detectable through microscopy.
Conclusions:
Our results suggest that Nanopore sequencing data of cfDNA from CSF samples may be a promising approach for initial brain tumor diagnostics and an important tool for disease monitoring.
Insights
Liquid biopsies using Nanopore sequencing of cell-free DNA from cerebrospinal fluid offer a less invasive method for brain tumor diagnosis and monitoring. This technique detects circulating tumor DNA, aiding in disease assessment without repeated tissue biopsies.
Area of Science:
- Neuro-oncology
- Genomics
- Molecular Diagnostics
Background:
- Molecular brain tumor diagnosis typically requires invasive tissue biopsies, posing risks especially for deep-seated lesions.
- Re-biopsies are often necessary to track tumor progression, recurrence, or genetic changes.
Purpose of the Study:
- To evaluate Nanopore sequencing of cell-free DNA (cfDNA) from cerebrospinal fluid (CSF) for brain tumor diagnostics and monitoring.
- To assess the utility of copy number variations (CNV) and DNA methylation profiling in CSF cfDNA for molecular tumor characterization.
Main Methods:
- Nanopore sequencing was performed on cfDNA extracted from CSF samples of 99 patients with 22 different brain tumor entities.
- Copy number variations (CNV) and global DNA methylation patterns were analyzed.
- A random forest classifier was used, and results were compared to clinical diagnosis and tumor tissue molecular analysis.
Main Results:
- 110 out of 129 samples were successfully sequenced, with 50 showing detectable circulating tumor DNA (ctDNA).
- CNV analysis identified diagnostic and prognostic alterations, often mirroring tumor tissue profiles.
- DNA methylation profiling enabled accurate tumor classification in 22 cases.
Conclusions:
- Nanopore sequencing of CSF cfDNA shows promise for non-invasive initial brain tumor diagnosis.
- This approach can serve as a valuable tool for monitoring disease progression and treatment response.
