The influence of biological and lifestyle factors on circulating cell-free DNA in blood plasma

Research and clinical use of circulating cell-free DNA (cirDNA) is expanding rapidly; however, there remain large gaps in our understanding of the influence of lifestyle and biological factors on the amount of cirDNA present in blood. Here, we review 66 individual studies of cirDNA levels and lifestyle and biological factors, including exercise (acute and chronic), alcohol consumption, occupational hazard exposure, smoking, body mass index, menstruation, hypertension, circadian rhythm, stress, biological sex and age. Despite technical and methodological inconsistences across studies, we identify acute exercise as a significant influence on cirDNA levels. Given the large increase in cirDNA induced by acute exercise, we recommend that controlling for physical activity prior to blood collection is routinely incorporated into study design when total cirDNA levels are of interest. We also highlight appropriate selection and complete reporting of laboratory protocols as important for improving the reproducibility cirDNA studies and ability to critically evaluate the results.

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Multicenter Evaluation of Circulating Cell-Free DNA Extraction and Downstream Analyses for the Development of Standardized (Pre)analytical Work Flows

Clinical Chemistry ◽

10.1373/clinchem.2019.306837 ◽

2019 ◽

Vol 66 (1) ◽

pp. 149-160 ◽

Cited By ~ 17

Author(s):

Rita Lampignano ◽

Martin H.D Neumann ◽

Sabrina Weber ◽

Vera Kloten ◽

Andrei Herdean ◽

...

Keyword(s):

Clinical Decision Making ◽

Clinical Decision ◽

Digital Pcr ◽

Blood Collection ◽

Routine Practice ◽

Acid Extraction ◽

Cell Free Dna ◽

Free Dna ◽

Analytical Work ◽

Circulating Cell Free Dna

Abstract BACKGROUND In cancer patients, circulating cell-free DNA (ccfDNA) can contain tumor-derived DNA (ctDNA), which enables noninvasive diagnosis, real-time monitoring, and treatment susceptibility testing. However, ctDNA fractions are highly variable, which challenges downstream applications. Therefore, established preanalytical work flows in combination with cost-efficient and reproducible reference materials for ccfDNA analyses are crucial for analytical validity and subsequently for clinical decision-making. METHODS We describe the efforts of the Innovative Medicines Initiative consortium CANCER-ID (http://www.cancer-id.eu) for comparing different technologies for ccfDNA purification, quantification, and characterization in a multicenter setting. To this end, in-house generated mononucleosomal DNA (mnDNA) from lung cancer cell lines carrying known TP53 mutations was spiked in pools of plasma from healthy donors generated from 2 different blood collection tubes (BCTs). ccfDNA extraction was performed at 15 partner sites according to their respective routine practice. Downstream analysis of ccfDNA with respect to recovery, integrity, and mutation analysis was performed centralized at 4 different sites. RESULTS We demonstrate suitability of mnDNA as a surrogate for ccfDNA as a process quality control from nucleic acid extraction to mutation detection. Although automated extraction protocols and quantitative PCR-based quantification methods yielded the most consistent and precise results, some kits preferentially recovered spiked mnDNA over endogenous ccfDNA. Mutated TP53 fragments derived from mnDNA were consistently detected using both next-generation sequencing-based deep sequencing and droplet digital PCR independently of BCT. CONCLUSIONS This comprehensive multicenter comparison of ccfDNA preanalytical and analytical work flows is an important contribution to establishing evidence-based guidelines for clinically feasible (pre)analytical work flows.

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Detection of EGFR alterations in circulating cell-free DNA of lung tumor patients: Higher sensitivity obtained with plasma compared to serum.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e19046 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e19046-e19046

Author(s):

Marc G. Denis ◽

Marie Marcq ◽

Paul Hofman ◽

Acya Bizieux-Thaminy ◽

Jaafar Bennouna ◽

...

Keyword(s):

Egfr Mutation ◽

Lung Tumor ◽

Blood Collection ◽

Molecular Testing ◽

Alternative Source ◽

Cell Free Dna ◽

Free Dna ◽

Tki Treatment ◽

Nsclc Patients ◽

Circulating Cell Free Dna

e19046 Background: Detection of EGFR alterations is critical for predicting the response to tyrosine kinase inhibitors (TKI) in patients with non-small-cell lung cancer (NSCLC). In clinical practice, molecular testing is performed on tumor tissues when available. We investigated the use of circulating cell-free DNA for the detection of EGFR alterations in patients with NSCLC. Methods: Serum and plasma were obtained by centrifugation (10 min, 2,000 g, 20°C) performed within 3 hours following blood collection. Cell-free DNA was extracted using the QIAamp Circulating Nucleic Acid kit (Qiagen). Detection of EGFR alterations was performed using the approved Therascreen EGFR RGQ kit (Qiagen). Samples were tested positive for EGFR mutation when the ΔCt value (Ct of the mutation specific PCR – Ct of the control PCR) was lower than 12 for exon 19 deletions, and below 14 for L858R mutation. Results: No EGFR alteration was detected in samples collected from healthy donors (n=6), NSCLC patients with a wild type EGFR (n=60), and early stages NSCLC patients presenting an EGFR mutation in their tumor (n=11). Thirteen metastatic patients presenting an EGFR mutation in their tumor were tested before initiation of TKI treatment. When available, both serum and plasma were analyzed. Nine of these patients (9/13; 69.2%) were tested positive in their serum. The ΔCt values obtained were lower for plasma than for serum in most cases, and more patients (10/11; 90.9%) were EGFR mutation positive when plasma was tested. Finally we tested 3 patients during TKI treatment on a monthly basis. For 2 patients we were unable to detect the mutation initially found in pretreatment samples. Clinically, both patients were partial responders. The third patient did not respond to TKI, and we detected the EGFR mutation with stable ΔCt values at all points tested. Conclusions: EGFR alterations can be found in patients presenting an EGFR mutation in a metastatic NSCLC. Plasma samples allowed a better detection rate. Our results suggest that DNA circulating in plasma is a useful alternative source of tumor DNA that could be used for determining EGFR mutation status, and for follow-up of treatment. Supported by a grant from Astra-Zeneca.

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Plasma free DNA

Biochemia Medica ◽

10.11613/bm.2019.010904 ◽

2018 ◽

Vol 29 (1) ◽

pp. 153-156 ◽

Cited By ~ 6

Author(s):

Dietmar Enko ◽

Gabriele Halwachs-Baumann ◽

Gernot Kriegshäuser

Keyword(s):

Blood Collection ◽

Plasma Samples ◽

Cell Free Dna ◽

Free Dna ◽

Roche Diagnostics Gmbh ◽

Dna Collection ◽

Roche Diagnostics ◽

The Impact ◽

Time Point ◽

Circulating Cell Free Dna

Introduction: Standardized pre-analytical blood sample procedures for the analysis of circulating cell-free DNA (ccfDNA) are still not available. Therefore, the present study aimed at evaluating the impact of storage conditions related to different times (24 and 48 h) and temperatures (room temperature (RT) and 4 - 8 °C) on the plasma ccfDNA concentration of blood samples drawn into Cell-Free DNA collection tubes (Roche Diagnostics GmbH, Mannheim, Germany). Materials and methods: Venous blood from 30 healthy individuals was collected into five 8.5 mL Cell-Free DNA Collection Tubes (Roche Diagnostics GmbH) each. Plasma samples were processed at time point of blood collection (tube 1), and after storage under the following conditions: 24 h at RT (tube 2) or 4-8 °C (tube 3), and 48 h at RT (tube 4) or 4 - 8 °C (tube 5). Circulating cell-free DNA concentrations were determined by EvaGreen chemistry-based droplet digital PCR (ddPCR). Results: No statistically significant differences between median (interquartile range) plasma ccfDNA concentrations (ng/mL) at time point of blood collection (3.17 (2.13 – 3.76)) and after storage for 24 h (RT: 3.02 (2.41 – 3.68); 4-8 °C: 3.21 (2.19 – 3.46)) and 48 h (RT: 3.13 (2.10 – 3.76); 4-8 °C: 3.09 (2.19 – 3.50)) were observed (P values from 0.102 – 0.975). Conclusions: No unwanted release of genomic DNA from white blood cells could be detected in plasma samples after tube storage for 24 and 48 h regardless of storage temperature.

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Sensitivity assessment of workflows detecting rare circulating cell-free DNA targets: A study design proposal

PLoS ONE ◽

10.1371/journal.pone.0253401 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0253401

Author(s):

Thorsten Voss ◽

Andrea Ullius ◽

Maike Schönborn ◽

Uwe Oelmüller

Keyword(s):

Study Design ◽

Liquid Biopsy ◽

Limit Of Detection ◽

Test Procedure ◽

Blood Collection ◽

Assay Sensitivity ◽

Cell Free Dna ◽

Free Dna ◽

Sensitivity Assessment ◽

Circulating Cell Free Dna

The field of liquid biopsy has seen extensive growth in recent decades, making it one of the most promising areas in molecular diagnostics. Circulating cell-free DNA (ccfDNA) especially is used as an analyte in a growing number of diagnostic assays. These assays require specified preanalytical workflows delivering ccfDNA in qualities and quantities that facilitate correct and reliable results. As each step and component used in the preanalytical process has the potential to influence the assay sensitivity and other performance characteristics, it is key to find an unbiased experimental setup to test these factors in diagnostic or research laboratories. We defined one such setup by using blood from healthy subjects and commercially available products for blood collection, spike-in material, ccfDNA isolation, and qPCR assays. As the primary read-out, we calculated the probit model-based LOD95 (limit of detection of the 95th percentile) from the qPCR assay results. In a proof of principle study we tested two different but widely used blood ccfDNA profile stabilization technologies in blood collection tubes, the Cell-Free DNA BCT and the PAXgene Blood ccfDNA Tube. We tested assays for three different EGFR gene mutations and one BRAF gene mutation. The study design revealed differences in performance between the two tested technologies for all four mutations. In conclusion, we successfully established a blueprint for a test procedure capable of verifying and validating a liquid biopsy workflow from blood collection to the analytical result.

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