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

ABSTRACT: The methylation pattern of cfDNA, isolated from liquid biopsies, is gaining substantial interest for diagnosis and monitoring of diseases. We have evaluated the impact of type of blood collection tube and time delay between blood draw and plasma preparation on bisulphite-based cfDNA methylation profiling. Fifteen tubes of blood were drawn from three healthy volunteer subjects (BD Vacutainer K2E EDTA spray tubes, Streck Cell-Free DNA BCT tubes, PAXgene Blood ccfDNA tubes, Roche Cell-Free DNA Collection tubes and Biomatrica LBgard blood tubes in triplicate). Samples were either immediately processed or stored at room temperature for 24 or 72 hours before plasma preparation. DNA fragment size was evaluated by capillary electrophoresis. Reduced representation bisulphite sequencing was performed on the cell-free DNA isolated from these plasma samples. We evaluated the impact of blood tube and time delay on several quality control metrics. All preservation tubes performed similar on the quality metrics that were evaluated. Furthermore, a considerable increase in cfDNA concentration and the fraction of it derived from NK cells was observed after a 72-hour time delay in EDTA tubes. The methylation pattern of cfDNA is robust and reproducible in between the different preservation tubes. EDTA tubes processed as soon as possible, preferably within 24 hours, are the most cost effective. If immediate processing is not possible, preservation tubes are valid alternatives.

SUBMITTER: Van Paemel R

PROVIDER: S-EPMC8216177 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionPrecision medicine has revolutionized the understanding and treatment of cancer by identifying subsets of patients who are amenable to specific treatments according to their molecular characteristics, as exemplified by epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC). Although tissue biopsy is the gold standard for determining molecular alterations in tumors, its limitations have prompted the development of new techniques for studying tumor biomarkers in liquid biopsies, such as mutation analysis in cell-free DNA (cfDNA). cfDNA analysis can accurately determine tumor progression and prognosis and more effectively identify appropriate targeted therapies. However, cfDNA is vulnerable, particularly during plasma sample shipping.ObjectiveWe compared the cell- and DNA-stabilizing properties of cell-free DNA blood collection tubes (BCTs) with those of the traditional shipping method (frozen plasma) for EGFR mutation testing using the cobas® EGFR Mutation Test v2 in a prospective cohort of 49 patients from three different Spanish hospitals.MethodsIn total, 98 NSCLC samples, two from each patient, were studied; five of the 49 cases were considered invalid by cobas® with one of the two shipping methods analyzed. After excluding these samples, we analyzed 88 samples from 44 patients. Considering the current methodology (frozen plasma) for sending samples as the gold standard, we evaluated the sensitivity and specificity of cfDNA BCT shipment.ResultsThe global agreement between the two methods was 95.4%, with 100% sensitivity and 94.6% specificity for the cfDNA BCTs. cfDNA BCTs had a positive predictive value of 81.8% and negative predictive value of 100%.ConclusioncfDNA BCTs have the same sensitivity for EGFR mutation analysis in liquid biopsy as the current methodology and very high specificity. They also have some additional advantages in terms of collection and further shipment. Therefore, cfDNA BCTs can be perfectly incorporated into the routine practice for EGFR mutation determination.FundingRoche Farma S.A., Spain.

Project description:Acute coronary syndrome (ACS) is a common disease with high mortality and morbidity rates. The methylation status of blood DNA may serve as a potential early diagnosis and prevention biomarker for numerous diseases. The present study was designed to explore novel genome-wide aberrant DNA methylation patterns associated with ACS. The Infinium HumanMethylation450 assay was used to examine genome-wide DNA methylation profiles in 3 pairs of ACS and control group samples. Epigenome-wide DNA methylation, genomic distribution, Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. The results were confirmed using methylation-specific polymerase chain reaction (MSP) and Sequenom MassARRAY analyses in ACS, stable coronary artery disease (SCAD) and control samples. A total of 11,342 differentially methylated (DM) 5'-C-phosphate-G-3' (CpG) sites were identified, including 8,865 hypomethylated and 2,477 hypermethylated CpG sites in the ACS group compared with the control samples. They varied in frequency across genomic compartments, but were particularly notable in gene bodies and shores. The results of GO term and KEGG pathway enrichment analyses revealed that the methylated genes were associated with certain biological processes and pathways. Despite the considerable variability in methylation data, the candidate selected possessed significant methylation alteration in mothers against decapentaplegic homolog 3 (SMAD3) transcription start site 155 (Chr1:67356838-Chr1:67356942). MSP analysis from 81 ACS samples, 74 SCAD samples and 53 healthy samples, and Sequenom MassARRAY analysis, confirmed that differential CpG methylation of SMAD3 was significantly corrected with the reference results of the HumanMethylation450 array. The data identified an ACS-specific DNA methylation profile with a large number of novel DM CpG sites, some of which may serve as candidate markers for the early diagnosis of ACS.

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

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