Direct circulating tumor DNA detection from unpurified plasma using a digital PCR platform

Sefrioui, David; Beaussire, Ludivine; Perdrix, A.; Clatot, Florian; Michel, Pierre; Frébourg, Thierry; Fiore, Frédéric Di; Sarafan-Vasseur, Nasrin

doi:10.1016/j.clinbiochem.2017.06.005

Cited by 11 publications

(6 citation statements)

References 13 publications

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“…The presence of contamination in cfDNA can, however, be accounted for by sequencing white blood cells and filtering somatic mutations attributable to clonal haematopoiesis [73], although this approach will not neutralise the diluting effect of such contamination on the tumour fraction of cfDNA. The necessity of extracting cfDNA from plasma was challenged by a study conducted by Sefrioui et al, The group compared concordance rates in a cohort of 17 mCRC patients and established 93% and 88% mutation detection rates for cfDNA isolated from plasma and crude plasma samples, respectively, suggesting that extraction of cfDNA from plasma may enhance detection by increasing tumour purity [74]. Similarly, increasing cfDNA concentration may also enhance concordance rates, by increasing the tumour fraction [75,76].…”

Section: Tumour Fraction and Mutation Allele Frequency (Maf)mentioning

confidence: 99%

Assessing the Concordance of Genomic Alterations between Circulating-Free DNA and Tumour Tissue in Cancer Patients

Jahangiri

Hurst

2019

Cancers

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Somatic alterations to the genomes of solid tumours, which in some cases represent actionable drivers, provide diagnostic and prognostic insight into these complex diseases. Spatial and longitudinal tracking of somatic genomic alterations (SGAs) in patient tumours has emerged as a new avenue of investigation, not only as a disease monitoring strategy, but also to improve our understanding of heterogeneity and clonal evolution from diagnosis through disease progression. Furthermore, analysis of circulating-free DNA (cfDNA) in the so-called “liquid biopsy” has emerged as a non-invasive method to identify genomic information to inform targeted therapy and may also capture the heterogeneity of the primary and metastatic tumours. Considering the potential of cfDNA analysis as a translational laboratory tool in clinical practice, establishing the extent to which cfDNA represents the SGAs of tumours, particularly actionable driver alterations, becomes a matter of importance, warranting standardisation of methods and practices. Here, we assess the utilisation of cfDNA for molecular profiling of SGAs in tumour tissue across a broad range of solid tumours. Moreover, we examine the underlying factors contributing to discordance of detected SGAs between cfDNA and tumour tissue.

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Section: Tumour Fraction and Mutation Allele Frequency (Maf)mentioning

confidence: 99%

Assessing the Concordance of Genomic Alterations between Circulating-Free DNA and Tumour Tissue in Cancer Patients

Jahangiri

Hurst

2019

Cancers

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“…To our knowledge, there are only a few related studies that reported nucleosomal DNA evaluation using dPCR directly from unpurified plasma. 24 To determine the copy number difference of both mutation and wild-type bearing DNAs, evaluations were conducted for genomic DNA (gDNA) and nDNA, which are similar in size to cell-free DNA. In summary, the size of the DNA is a critical factor in measuring absolute copy number according to our tests with various concentrations.…”

Section: Discussionmentioning

confidence: 99%

“…However, there are only a few research reports that have directly tested the matrix effect. In one pilot study, which aimed to evaluate ctDNA detection using the dPCR platform, ctDNA was detected in metastatic colorectal cancer (mCRC) patients directly from plasma as well as after an isolation step 24 . In this study, we conclude that optimized conditions are required to increase the precision of ddPCR to develop reference materials with matrix conditions.…”

Section: Introductionmentioning

confidence: 99%

Improvement of digital PCR conditions for direct detection of KRAS mutations

Lee

Kim

Kang

et al. 2020

Clinical Laboratory Analysis

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Background In standard analytical conditions, an isolation step is essential for circulating tumor DNA (ctDNA) analysis. The necessity of this step becomes unclear with the development of highly sensitive detection methods. The aim of this study was to evaluate ctDNA mimetic nDNA detection as reference materials (RMs) using dPCR technologies either directly from serum or without serum. Methods To determine an absolute count of both mutation and wild‐type bearing DNA molecules, genomic DNA (gDNA) and nucleosomal DNA (nDNA), which are similar in size to cell‐free DNA, were evaluated. We tested 3 KRAS mutations in colorectal cancer cell lines. Results We describe the recent progress in RMs. The short DNA fragments, such as sDNA and nDNA, exhibited higher quantitative values of dPCR compared to gDNA. The efficiency between Atlantis dsDNase (AD) and Micrococcal Nuclease (MN) affects DNA quantification. Moreover, there was a significant difference in dPCR output when spiking gDNA or nDNA containing KRAS mutations into FBS compared to the dPCR output under non‐FBS conditions. Conclusion The matrix effect crucially affects the accuracy of gDNA and nDNA level estimation in the direct detection of mimic of patient samples. The form of reference material we proposed should be optimized for various conditions to develop reference materials that can accurately measure copy number and verify the detection of KRAS mutations in the matrix.

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“…In addition, dPCR overcomes the shortcomings in qPCR, such as the need for the normalization of the target gene-of-interest to endogenous reference or housekeeping genes, and the need for extraction, both of which result in inaccurate quantification and hampers inter-laboratory comparability of data [22,23] [24]. While direct DNA quantification from plasma or serum using dPCR was previously shown [21,25–27], one step direct detection and RNA quantification in oocytes has not yet been successfully shown. Single oocyte places a physical limitation on RNA isolation due to its minute cellular and genetic volume, as well as the risk of loss.…”

Section: Introductionmentioning

confidence: 99%

“…In recent years, digital PCR (dPCR) has gained popularity in quantifying genetic material [17–19]. The technique of partitioning allows for high signal-to-noise ratio, high sensitivity to quantify absolute target genes, and being calibration free, are major advantages of dPCR over that of quantitative PCR (qPCR) [20,21]. In addition, dPCR overcomes the shortcomings in qPCR, such as the need for the normalization of the target gene-of-interest to endogenous reference or housekeeping genes, and the need for extraction, both of which result in inaccurate quantification and hampers inter-laboratory comparability of data [22,23] [24].…”

Section: Introductionmentioning

confidence: 99%

Digital PCR quantification of DNA, RNA and extracellular microRNA of mouse oocytes

et al. 2021

Preprint

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Despite numerous advances in in vitro fertilization (IVF) techniques since its first success in 1978, almost half of the patients treated remain childless. The multifactorial nature of IVF treatment means that success is dependent on variables, including the quality of oocytes. Therefore, new technologies are needed to objectively and quantitatively examine how each oocyte can be selected or optimized to achieve for the best possible outcomes for patients. Here, we report an optimized digital polymerase chain reaction (dPCR) for direct absolute quantification of nucleic acids within 3.5 h without the need for sample extraction or purification. Using individual oocytes, the developed method demonstrated absolute quantification with a linear dynamic range of 0.65 – 33 copies/µL (r2=0.999), high accuracy and excellent reproducibility of <10% relative standard deviation. The method then identified the variable expression of Gapdh (0.72–16.95 copies/oocyte), Hprt1 (1.05–19.05 copies/oocyte) and ATPase 6, (5.55–32358.15 copies/oocyte) in ovaries even from the same mouse. Finally, dPCR was used to validate extracellular microRNAs from oocytes incubated with a toxic unsaturated very-long chained ceramide. This study therefore shows the feasibility of dPCR for the rapid and sensitive absolute quantification of DNA/RNA and extracellular miRNA for the study of oocytes.

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Direct circulating tumor DNA detection from unpurified plasma using a digital PCR platform

Cited by 11 publications

References 13 publications

Assessing the Concordance of Genomic Alterations between Circulating-Free DNA and Tumour Tissue in Cancer Patients

Assessing the Concordance of Genomic Alterations between Circulating-Free DNA and Tumour Tissue in Cancer Patients

Improvement of digital PCR conditions for direct detection of KRAS mutations

Digital PCR quantification of DNA, RNA and extracellular microRNA of mouse oocytes

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