scholarly journals High throughput and affordable genome-wide methylation profiling of circulating cell-free DNA by Methylated DNA sequencing (MeD-seq) of LpnPI digested fragments

2021 ◽  
Author(s):  
Teoman Deger ◽  
Ruben G Boers ◽  
Vanja de Weerd ◽  
Lindsay Angus ◽  
Marjolijn MJ van der Put ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cancer Patients ◽  
Blood Collection ◽  
Liquid Biopsies ◽  
Free Dna ◽  
Genome Wide ◽  
Isolation Methods ◽  
Load Monitoring ◽  
Blood Collection Tubes ◽  
Methylation Profiling

Background: DNA methylation detection in liquid biopsies provides a highly promising and much needed means for real-time monitoring of disease load in advanced cancer patient care. Compared to the often-used somatic mutations, tissue- and cancer-type specific epigenetic marks affect a larger part of the cancer genome and generally have a high penetrance throughout the tumour. Here we describe the successful application of the recently described MeD-seq assay for genome-wide DNA methylation profiling on cell-free DNA (cfDNA). The compatibility of the MeD-seq assay with different types of blood collection tubes, cfDNA input amounts, cfDNA isolation methods, and vacuum-concentration of samples was evaluated using plasma from both metastatic cancer patients and healthy blood donors (HBDs). To investigate the potential value of cfDNA methylation profiling for tumour load monitoring, we profiled paired samples from 8 patients with resectable colorectal liver metastases (CRLM) before and after surgery. Results: The MeD-seq assay worked on plasma-derived cfDNA from both EDTA and CellSave blood collection tubes when at least 10 ng of cfDNA was used. From the 3 evaluated cfDNA isolation methods, both the manual QIAamp Circulating Nucleic Acid Kit (Qiagen) and the semi-automated Maxwell RSC ccfDNA Plasma Kit (Promega) were compatible with MeD-seq analysis, whereas the QIAsymphony DSP Circulating DNA Kit (Qiagen) yielded significantly fewer reads when compared to the QIAamp kit (P<0.001). Vacuum-concentration of samples before MeD-seq analysis was possible with samples in AVE buffer (QIAamp) or water, but yielded inconsistent results for samples in EDTA-containing Maxwell buffer. Principal component analysis showed that pre-surgical samples from CRLM patients were very distinct from HBDs, whereas post-surgical samples were more similar. Several described methylation markers for colorectal cancer monitoring in liquid biopsies showed differential methylation between pre-surgical CRLM samples and HBDs in our data, supporting the validity of our approach. Results for MSC, ITGA4, GRIA4, and EYA4, were validated by quantitative methylation specific PCR. Conclusions: The MeD-seq assay provides a promising new method for cfDNA methylation profiling. Potential future applications of the assay include marker discovery specifically for liquid biopsy analysis as well as direct use as a disease load monitoring tool in advanced cancer patients.

scholarly journals High-throughput and affordable genome-wide methylation profiling of circulating cell-free DNA by methylated DNA sequencing (MeD-seq) of LpnPI digested fragments

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Teoman Deger ◽  
Ruben G. Boers ◽  
Vanja de Weerd ◽  
Lindsay Angus ◽  
Marjolijn M. J. van der Put ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cancer Patients ◽  
Blood Collection ◽  
Liquid Biopsies ◽  
Free Dna ◽  
Genome Wide ◽  
Isolation Methods ◽  
Load Monitoring ◽  
Blood Collection Tubes ◽  
Methylation Profiling

Abstract Background DNA methylation detection in liquid biopsies provides a highly promising and much needed means for real-time monitoring of disease load in advanced cancer patient care. Compared to the often-used somatic mutations, tissue- and cancer-type specific epigenetic marks affect a larger part of the cancer genome and generally have a high penetrance throughout the tumour. Here, we describe the successful application of the recently described MeD-seq assay for genome-wide DNA methylation profiling on cell-free DNA (cfDNA). The compatibility of the MeD-seq assay with different types of blood collection tubes, cfDNA input amounts, cfDNA isolation methods, and vacuum concentration of samples was evaluated using plasma from both metastatic cancer patients and healthy blood donors (HBDs). To investigate the potential value of cfDNA methylation profiling for tumour load monitoring, we profiled paired samples from 8 patients with resectable colorectal liver metastases (CRLM) before and after surgery. Results The MeD-seq assay worked on plasma-derived cfDNA from both EDTA and CellSave blood collection tubes when at least 10 ng of cfDNA was used. From the 3 evaluated cfDNA isolation methods, both the manual QIAamp Circulating Nucleic Acid Kit (Qiagen) and the semi-automated Maxwell® RSC ccfDNA Plasma Kit (Promega) were compatible with MeD-seq analysis, whereas the QiaSymphony DSP Circulating DNA Kit (Qiagen) yielded significantly fewer reads when compared to the QIAamp kit (p < 0.001). Vacuum concentration of samples before MeD-seq analysis was possible with samples in AVE buffer (QIAamp) or water, but yielded inconsistent results for samples in EDTA-containing Maxwell buffer. Principal component analysis showed that pre-surgical samples from CRLM patients were very distinct from HBDs, whereas post-surgical samples were more similar. Several described methylation markers for colorectal cancer monitoring in liquid biopsies showed differential methylation between pre-surgical CRLM samples and HBDs in our data, supporting the validity of our approach. Results for MSC, ITGA4, GRIA4, and EYA4 were validated by quantitative methylation specific PCR. Conclusions The MeD-seq assay provides a promising new method for cfDNA methylation profiling. Potential future applications of the assay include marker discovery specifically for liquid biopsy analysis as well as direct use as a disease load monitoring tool in advanced cancer patients.

scholarly journals Genome-Wide Analysis of Cell-Free DNA Methylation Profiling with MeDIP-Seq Identified Potential Biomarkers for Colorectal Cancer

2021 ◽  
Author(s):  
Xin Zhang ◽  
Tao Li ◽  
Qiang Niu ◽  
Chang Jiang Qin ◽  
Ming Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
Cancer Patients ◽  
Cell Free Dna ◽  
Free Dna ◽  
Genome Wide ◽  
A Genome ◽  
Colorectal Cancer Patients ◽  
Methylation Profiling ◽  
Potential Biomarkers

Abstract Background: To verify the feasibility of genome-wide plasma cell-free DNA(cfDNA) methylation profiling for early diagnosis of colorectal cancer.Methods: We performed a genome-wide cfDNA methylation profiling study of colorectal cancer patients by methylated DNA immunoprecipitation coupled with high throughput sequencing (MeDIP-seq).Results: Compared with the control group, 939 differentially methylated regions (DMRs) located in promoter regions were found in colorectal cancer patients, 16 of these DMRs were hypermethylated and the remaining 923 were hypomethylated. In addition, these hypermethylated genes, mainly including PRDM14, RALYL, ELMOD1, and TMEM132E, were validated and confirmed in colorectal cancer by using publicly available DNA methylation data.Conclusions: Our study indicates that MeDIP-seq can be used as an optimal approach for analyzing cfDNA methylomes, and the differentially methylated genes obtained by MeDIP-seq can be used as potential biomarkers for clinical application in patients with colorectal cancer.

scholarly journals Genome-Wide Plasma Cell-Free DNA Methylation Profiling Identifies Potential Biomarkers for Lung Cancer

2019 ◽  
Vol 2019 ◽  
pp. 1-7 ◽  
Author(s):  
Wei Xu ◽  
Jun Lu ◽  
Qiang Zhao ◽  
Jun Wu ◽  
Jielin Sun ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Dna Methylation ◽  
Cancer Patients ◽  
Gene Promoters ◽  
Cell Free Dna ◽  
Lung Cancer Patients ◽  
Free Dna ◽  
Differentially Methylated Genes ◽  
Potential Biomarker ◽  
Methylation Profiling

As a noninvasive blood testing, the detection of cell-free DNA (cfDNA) methylation in plasma has raised an increasing interest due to diagnostic applications. Although extensively used in cfDNA methylation analysis, bisulfite sequencing is less cost-effective. In this study, we investigated the cfDNA methylation patterns in lung cancer patients by MeDIP-seq. Compared with the healthy individuals, 330 differentially methylated regions (DMRs) at gene promoters were identified in lung cancer patients with 33 hypermethylated and 297 hypomethylated regions, respectively. Moreover, these hypermethylated genes were validated with the publicly available DNA methylation data, yielding a set of ten significant differentially methylated genes in lung cancer, including B3GAT2, BCAR1, HLF, HOPX, HOXD11, MIR1203, MYL9, SLC9A3R2, SYT5, and VTRNA1-3. Our study demonstrated MeDIP-seq could be effectively used for cfDNA methylation profiling and identified a set of potential biomarker genes with clinical application for lung cancer.

scholarly journals Genome-wide study of the effect of blood collection tubes on the cell-free DNA methylome

Epigenetics ◽  
2020 ◽  
pp. 1-11
Author(s):  
Ruben Van Paemel ◽  
Andries De Koker ◽  
Christa Caggiano ◽  
Annelien Morlion ◽  
Pieter Mestdagh ◽  
...  
Keyword(s):  
Blood Collection ◽  
Cell Free Dna ◽  
Dna Methylome ◽  
Free Dna ◽  
Genome Wide ◽  
Blood Collection Tubes ◽  
Genome Wide Study

scholarly journals Aging-associated distinctive DNA methylation changes of LINE-1 retrotransposons in pure cell-free DNA from human blood

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Wardah Mahmood ◽  
Lars Erichsen ◽  
Pauline Ott ◽  
Wolfgang A. Schulz ◽  
Johannes C. Fischer ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Free Dna ◽  
The Past ◽  
Free Dna ◽  
Genome Wide ◽  
Cancerous Cell ◽  
Pure Cell ◽  
Epigenetic Biomarker ◽  
Methylation Patterns ◽  
Cancerous Cells

AbstractLINE-1 hypomethylation of cell-free DNA has been described as an epigenetic biomarker of human aging. However, in the past, insufficient differentiation between cellular and cell-free DNA may have confounded analyses of genome-wide methylation levels in aging cells. Here we present a new methodological strategy to properly and unambiguously extract DNA methylation patterns of repetitive, as well as single genetic loci from pure cell-free DNA from peripheral blood. Since this nucleic acid fraction originates mainly in apoptotic, senescent and cancerous cells, this approach allows efficient analysis of aged and cancerous cell-specific DNA methylation patterns for diagnostic and prognostic purposes. Using this methodology, we observe a significant age-associated erosion of LINE-1 methylation in cfDNA suggesting that the threshold of hypomethylation sufficient for relevant LINE-1 activation and consequential harmful retrotransposition might be reached at higher age. We speculate that this process might contribute to making aging the main risk factor for many cancers.

Clinical Relevance of Plasma DNA Methylation in Colorectal Cancer Patients Identified by Using a Genome-Wide High-Resolution Array

2014 ◽  
Vol 22 (S3) ◽  
pp. 1419-1427 ◽  
Author(s):  
Pei-Ching Lin ◽  
Jen-Kou Lin ◽  
Chien-Hsing Lin ◽  
Hung-Hsin Lin ◽  
Shung-Haur Yang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Dna Methylation ◽  
High Resolution ◽  
Cancer Patients ◽  
Clinical Relevance ◽  
Plasma Dna ◽  
Genome Wide ◽  
A Genome ◽  
Colorectal Cancer Patients

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

2016 ◽  
Vol 8 (1) ◽  
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

scholarly journals Comparison of Blood Collection Tubes from Three Different Manufacturers for the Collection of Cell-Free DNA for Liquid Biopsy Mutation Testing

2017 ◽  
Vol 19 (5) ◽  
pp. 801-804 ◽  
Author(s):  
Christina Alidousty ◽  
Danielle Brandes ◽  
Carina Heydt ◽  
Svenja Wagener ◽  
Maike Wittersheim ◽  
...  
Keyword(s):  
Liquid Biopsy ◽  
Mutation Testing ◽  
Blood Collection ◽  
Cell Free Dna ◽  
Free Dna ◽  
Blood Collection Tubes

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

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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