Article
Deciphering the methylation signature of circulating extracellular vesicle DNA for CNS tumour classification
Entschlüsselung der Methylierungssignatur von zirkulierender extrazellulärer Vesikel-DNA zur Klassifizierung von ZNS-Tumoren
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Published: | May 25, 2022 |
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Objective: Genome-wide methylation profiling has recently been developed into a tool that allows subtype tumor classification in central nervous system (CNS) tumors. We previously showed that extracellular vesicle (EV) DNA faithfully reflects the tumor methylation class, including information on the IDH mutation and MGMT promoter methylation status. Furthermore we showed that circulating plasma EVs are elevated in CNS tumor patients in comparison to healthy controls with tumor related protein profiles. In our ongoing work, we investigated, whether the methylation signatures of circulating EV DNA as well as cfDNA can be used for liquid biopsy approaches for CNS tumor detection and classification.
Methods: We isolated DNA from patients suffering from Glioblastoma (GBM), Meningioma (MGN) and cerebral metastases (CM) from circulating EVs (n=27), cfDNA (n=27) and tissue DNA (n=90). Patients undergoing epileptic surgery as well as aneurysm clipping were used as non-tumorous controls (HD, n= 7). EVs were classified by nanoparticle analysis (NTA), immunoblotting, imaging flow cytometry (IFCM) and electron microscopy.
Results: Isolated DNA showed higher molecular DNA in EV DNA in comparison to cfDNA, while HD or tumor patients showed not differences in their corresponding DNA size profiles. Next, we performed genome-wide methylation profiling by 850k Illumina EPIC array on all DNA analytes and tumor entities. Linear models and empirical Bayes methods identified significant differential methylated CPGs (GBM vs. HD, MGN, vs HD, CM vs. HD), that revealed tumor specific signatures to detect and discriminate different CNS tumor entities. Visualization of differential methylated CPGs by dimension reduction (PCA, t-SNE, Umap) verified tumor specific clusters. cfDNA and EV-DNA comparisons each revealed their own differential CPG profile.
Conclusion: Our study shows that the methylation signature of circulating EV DNA and cfDNA can be used to separate healthy individuals from tumor patients with the goal to augment standard-of-care imaging to improve tumor detection, classification and surveillance.