Genome-wide methylation profiling of glioblastoma extracellular vesicle DNA allows tumor classification

Abstract 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 non-tumor donors (HD) controls with tumor related protein profiles. We now investigated, whether the methylation signatures of circulating DNA (both EV and cfNDA) can be used in liquid biopsy approaches for CNS tumor detection and classification. We isolated DNA from circulating EVs (n=27), cfDNA (n=27) and tumor tissue DNA (n=90) of patients with glioblastoma (GBM), meningioma (MGN) and cerebral metastases (CM). Patients undergoing epilepsy surgery as well as aneurysm clipping were used as non-tumor controls (HD, n= 7). EVs were classified by nanoparticle analysis, immunoblotting, imaging flow cytometry and electron microscopy. Isolated EV-DNA comprised many sorts of molecular weight (up tp >10Kb) in comparison to cfDNA (130-140bp). Healthy donors and tumor patients showed not differences in their DNA size profiles. We performed genome-wide methylation profiling by 850k Illumina EPIC arrays for all DNA analytes and tumor entities. Linear models and empirical Bayes methods identified significant differentially 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 differentially methylated CPGs by dimension reduction (PCA, t-SNE, Umap) verified tumor specific clusters. cfDNA and EV-DNA exhibited distinctive individual CpG profiles. Our study shows that the methylation signature of circulating EV DNA and cfDNA can be used to separate healthy individuals from tumor patients and could potentially complement standard-of-care imaging to improve tumor detection, classification and surveillance.

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GENE-22. GENOME-WIDE METHYLATION PROFILING OF GLIOBLASTOMA EXTRACELLULAR VESICLE DNA ALLOWS TUMOR CLASSIFICATION

Neuro-Oncology ◽

10.1093/neuonc/noz175.424 ◽

2019 ◽

Vol 21 (Supplement_6) ◽

pp. vi102-vi102

Author(s):

Franz Ricklefs ◽

Cecile Maire ◽

Katharina Kolbe ◽

Mareike Holz ◽

Manfred Westphal ◽

...

Keyword(s):

Copy Number ◽

Methylation Status ◽

Extracellular Vesicle ◽

Copy Number Variations ◽

Tumor Classification ◽

Cns Tumors ◽

Original Tumor ◽

Genome Wide ◽

Potential Biomarker ◽

Methylation Profiling

Abstract BACKGROUND Genome-wide methylation profiling has recently been developed into a tool that allows subtype tumor classification in central nervous system (CNS) tumors. Extracellular vesicles (EVs) are released by CNS tumor cells and contain high molecular weight tumor DNA, rendering EVs a potential biomarker source to identify tumor subgroups, stratify patients and monitor therapy by liquid biopsy. We investigated whether the DNA in glioma-derived EVs reflects genome-wide tumor methylation profiles and allows tumor subtype classification. METHODS DNA was isolated from EVs secreted by cultured glioma stem-like cells (GSC) as well as from the cells of origin and from the original tumor samples (n=3 patients). EVs were classified by nanoparticle analysis (NTA), immunoblotting, imaging flow cytometry (IFCM), multiplex EV assays and electron microscopy. Genome-wide DNA methylation profiling was performed using an 850k Illumina EPIC array and results were classified according to the DKFZ brain tumor classifier. RESULTS The size range of GSC-derived EVs was 120–150 nm, as measured by NTA. The majority of secreted EVs exhibited high expression of common EV markers (i.e. CD9, CD63 and CD81), as characterized by IFCM and multiplex EV assays. Genome-wide methylation profiling of GSC-derived EVs correctly identified the methylation class of the original tumor, including information on the IDH mutation status and subclass classification (RTK1, RTK2). In addition, copy number alterations and the MGMT metyhlation status matched the pattern of the parental GSCs and original tumor samples. CONCLUSION EV DNA faithfully reflects the tumor methylation class as well as the MGMT methylation status and copy number variations present in the parental cells and the original tumor. Methylation profiling of circulating tumor EV DNA could become a useful tool to detect and classify CNS tumors.

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Genome-wide methylation profiling of glioblastoma cell-derived extracellular vesicle DNA allows tumor classification

Neuro-Oncology ◽

10.1093/neuonc/noab012 ◽

2021 ◽

Author(s):

Cecile L Maire ◽

Marceline M Fuh ◽

Kerstin Kaulich ◽

Krystian D Fita ◽

Ines Stevic ◽

...

Keyword(s):

Copy Number ◽

Methylation Status ◽

Tumor Classification ◽

Glioblastoma Cell ◽

Glioblastoma Cells ◽

High Molecular Weight Dna ◽

Genome Wide ◽

Tumor Identification ◽

Potential Biomarker ◽

Methylation Profiling

Abstract Background Genome-wide DNA methylation profiling has recently been developed into a tool that allows tumor classification in central nervous system tumors. Extracellular vesicles (EVs) are released by tumor cells and contain high molecular weight DNA, rendering EVs a potential biomarker source to identify tumor subgroups, stratify patients and monitor therapy by liquid biopsy. We investigated whether the DNA in glioblastoma cell-derived EVs reflects genome-wide tumor methylation and mutational profiles and allows non-invasive tumor subtype classification. Methods DNA was isolated from EVs secreted by glioblastoma cells as well as from matching cultured cells and tumors. EV-DNA was localized and quantified by direct stochastic optical reconstruction microscopy. Methylation and copy number profiling was performed using 850k arrays. Mutations were identified by targeted gene panel sequencing. Proteins were differentially quantified by mass spectrometric proteomics. Results Genome-wide methylation profiling of glioblastoma-derived EVs correctly identified the methylation class of the parental cells and original tumors, including the MGMT promoter methylation status. Tumor-specific mutations and copy number variations (CNV) were detected in EV-DNA with high accuracy. Different EV isolation techniques did not affect the methylation profiling and CNV results. DNA was present inside EVs and on the EV surface. Proteome analysis did not allow specific tumor identification or classification but identified tumor-associated proteins that could potentially be useful for enriching tumor-derived circulating EVs from biofluids. Conclusions This study provides proof of principle that EV-DNA reflects the genome-wide methylation, CNV and mutational status of glioblastoma cells and enables their molecular classification.

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Genome-wide DNA methylation profiling with MeDIP-seq using archived dried blood spots

Clinical Epigenetics ◽

10.1186/s13148-016-0242-1 ◽

2016 ◽

Vol 8 (1) ◽

Cited By ~ 22

Author(s):

Nicklas H. Staunstrup ◽

Anna Starnawska ◽

Mette Nyegaard ◽

Lene Christiansen ◽

Anders L. Nielsen ◽

...

Keyword(s):

Dna Methylation ◽

Dried Blood Spots ◽

Blood Spots ◽

Genome Wide ◽

Dna Methylation Profiling ◽

Methylation Profiling

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Integrative analysis reveals early and distinct genetic and epigenetic changes in intraductal papillary and tubulopapillary cholangiocarcinogenesis

Gut ◽

10.1136/gutjnl-2020-322983 ◽

2021 ◽

pp. gutjnl-2020-322983

Author(s):

Benjamin Goeppert ◽

Damian Stichel ◽

Reka Toth ◽

Sarah Fritzsche ◽

Moritz Anton Loeffler ◽

...

Keyword(s):

Treatment Options ◽

Genetic Alterations ◽

Integrative Analysis ◽

Intermediate Form ◽

Precursor Lesions ◽

Molecular Alterations ◽

Genome Wide ◽

Cells Of Origin ◽

Sample Set ◽

Methylation Profiling

ObjectiveA detailed understanding of the molecular alterations in different forms of cholangiocarcinogenesis is crucial for a better understanding of cholangiocarcinoma (CCA) and may pave the way to early diagnosis and better treatment options.DesignWe analysed a clinicopathologically well-characterised patient cohort (n=54) with high-grade intraductal papillary (IPNB) or tubulopapillary (ITPN) neoplastic precursor lesions of the biliary tract and correlated the results with an independent non-IPNB/ITPN associated CCA cohort (n=294). The triplet sample set of non-neoplastic biliary epithelium, precursor and invasive CCA was analysed by next generation sequencing, DNA copy number and genome-wide methylation profiling.ResultsPatients with invasive CCA arising from IPNB/ITPN had better prognosis than patients with CCA not associated with IPNB/ITPN. ITPN was localised mostly intrahepatic, whereas IPNB was mostly of extrahepatic origin. IPNB/ITPN were equally associated with small-duct and large-duct type intrahepatic CCA. IPNB exhibited mutational profiles of extrahepatic CCA, while ITPN had significantly fewer mutations. Most mutations were shared between precursor lesions and corresponding invasive CCA but ROBO2 mutations occurred exclusively in invasive CCA and CTNNB1 mutations were mainly present in precursor lesions. In addition, IPNB and ITPN differed in their DNA methylation profiles and analyses of latent methylation components suggested that IPNB and ITPN may have different cells-of-origin.ConclusionIntegrative analysis revealed that IPNB and ITPN harbour distinct early genetic alterations, IPNB are enriched in mutations typical for extrahepatic CCA, whereas ITPN exhibited few genetic alterations and showed distinct epigenetic profiles. In conclusion, IPNB/ITPN may represent a distinctive, intermediate form of intrahepatic and extrahepatic cholangiocarcinogenesis.

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EPCO-17. METHYLATION ANALYSIS OF MATCHED PRIMARY AND RECURRENT IDHmt ASTROCYTOMA; AN UPDATE FROM THE GLIOMA LONGITUDINAL ANALYSIS NL (GLASS-NL) CONSORTIUM

Neuro-Oncology ◽

10.1093/neuonc/noab196.016 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi5-vi5

Author(s):

Wies Vallentgoed ◽

Anneke Niers ◽

Karin van Garderen ◽

Martin van den Bent ◽

Kaspar Draaisma ◽

...

Keyword(s):

Dna Methylation ◽

Surgical Resection ◽

Molecular Mechanisms ◽

Relative Proportion ◽

Low Grade ◽

High Grade ◽

Primary Tumors ◽

Genome Wide ◽

Dna Methylation Profiling ◽

Methylation Profiling

Abstract The GLASS-NL consortium, was initiated to gain insight into the molecular mechanisms underlying glioma evolution and to identify markers of progression in IDH-mutant astrocytomas. Here, we present the first results of genome-wide DNA-methylation profiling of GLASS-NL samples. 110 adult patients were identified with an IDH-mutant astrocytoma at first diagnosis. All patients underwent a surgical resection of the tumor at least twice, separated by at least 6 months (median 40.9 months (IQR: 24.0, 64.7). In 37% and 18% of the cases, patients were treated with radiotherapy or chemotherapy respectively, before surgical resection of the recurrent tumor. DNA-methylation profiling was done on 235 samples from 103 patients (102 1st, 101 2nd, 29 3rd, and 3 4th resection). Copy number variations were also extracted from these data. Methylation classes were determined according to Capper et al. Overall survival (OS) was measured from date of first surgery. Of all primary tumors, the methylation-classifier assigned 85 (87%) to the low grade subclass and 10 (10%) to the high grade subclass. The relative proportion of high grade tumors increased ~three-fold at tumor recurrence (32/101, 32%) and even further in the second recurrence (15/29, 52%). Methylation classes were prognostic, both in primary and recurrent tumors. The overall DNA-methylation levels of recurrent samples was lower than that of primary samples. This difference is explained by the increased number of high grade samples at recurrence, since near identical DNA-methylation levels were observed in samples that remained low grade. In an unsupervised analysis, DNA-methylation data derived from primary and first recurrence samples of individual patients mostly (79%) cluster together. Recurrent samples that do not cluster with their primary tumor, form a separate group with relatively low genome-wide DNA-methylation. Our data demonstrate that methylation profiling identifies a shift towards a higher grade at tumor progression coinciding with reduced genome-wide DNA-methylation levels.

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Genome-wide DNA methylation profiling shows a distinct epigenetic signature associated with lung macrophages in cystic fibrosis

Clinical Epigenetics ◽

10.1186/s13148-018-0580-2 ◽

2018 ◽

Vol 10 (1) ◽

Cited By ~ 2

Author(s):

Youdinghuan Chen ◽

David A. Armstrong ◽

Lucas A. Salas ◽

Haley F. Hazlett ◽

Amanda B. Nymon ◽

...

Keyword(s):

Cystic Fibrosis ◽

Dna Methylation ◽

Lung Macrophages ◽

Genome Wide ◽

Dna Methylation Profiling ◽

Methylation Profiling

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Genome-wide interrogation of extracellular vesicle biology using barcoded miRNAs

eLife ◽

10.7554/elife.41460 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 7

Author(s):

Albert Lu ◽

Paulina Wawro ◽

David W Morgens ◽

Fernando Portela ◽

Michael C Bassik ◽

...

Keyword(s):

Nucleic Acid ◽

Molecular Mechanisms ◽

Membrane Vesicles ◽

Extracellular Vesicle ◽

Biologically Active ◽

Vesicle Release ◽

Guide Rnas ◽

Disease States ◽

Genome Wide ◽

First Time

Extracellular vesicles mediate transfer of biologically active molecules between neighboring or distant cells, and these vesicles may play important roles in normal physiology and the pathogenesis of multiple disease states including cancer. However, the underlying molecular mechanisms of their biogenesis and release remain unknown. We designed artificially barcoded, exosomal microRNAs (bEXOmiRs) to monitor extracellular vesicle release quantitatively using deep sequencing. We then expressed distinct pairs of CRISPR guide RNAs and bEXOmiRs, enabling identification of genes influencing bEXOmiR secretion from Cas9-edited cells. This approach uncovered genes with unrecognized roles in multivesicular endosome exocytosis, including critical roles for Wnt signaling in extracellular vesicle release regulation. Coupling bEXOmiR reporter analysis with CRISPR-Cas9 screening provides a powerful and unbiased means to study extracellular vesicle biology and for the first time, to associate a nucleic acid tag with individual membrane vesicles.

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