Cell-free DNA: Preanalytical variables

Bronkhorst, Abel J.; Aucamp, Janine; Pretorius, P. J.

doi:10.1016/j.cca.2015.08.028

Cited by 148 publications

(97 citation statements)

References 70 publications

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“…39), which has largely shaped efforts on optimization of the cfDNA isolation protocols. Although there is no clear consensus on the best practices for sample handling, we refer the reader to a review covering preanalytic variables that affect cfDNA quality (40). One key observation reported therein is that while overall, serum may yield higher levels of cfDNA than plasma, the yield is more variable and the cfDNA quality may be severely impacted due to lysis of monocytes.…”

Section: Collection and Processing Of Blood For Cfdnamentioning

confidence: 99%

Cell-free DNA (cfDNA): Clinical Significance and Utility in Cancer Shaped By Emerging Technologies

Volik¹,

Alcaide

Morin

et al. 2016

Molecular Cancer Research

295

235

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Precision oncology is predicated upon the ability to detect specific actionable genomic alterations and to monitor their adaptive evolution during treatment to counter resistance. Because of spatial and temporal heterogeneity and comorbidities associated with obtaining tumor tissues, especially in the case of metastatic disease, traditional methods for tumor sampling are impractical for this application. Known to be present in the blood of cancer patients for decades, cell-free DNA (cfDNA) is beginning to inform on tumor genetics, tumor burden, and mechanisms of progression and drug resistance. This substrate is amenable for inexpensive noninvasive testing and thus presents a viable approach to serial sampling for screening and monitoring tumor progression. The fragmentation, low yield, and variable admixture of normal DNA present formidable technical challenges for realization of this potential. This review summarizes the history of cfDNA discovery, its biological properties, and explores emerging technologies for clinically relevant sequence-based analysis of cfDNA in cancer patients. Molecular barcoding (or Unique Molecular Identifier, UMI)-based methods currently appear to offer an optimal balance between sensitivity, flexibility, and cost and constitute a promising approach for clinically relevant assays for near real-time monitoring of treatment-induced mutational adaptations to guide evidence-based precision oncology.

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Section: Collection and Processing Of Blood For Cfdnamentioning

confidence: 99%

Cell-free DNA (cfDNA): Clinical Significance and Utility in Cancer Shaped By Emerging Technologies

Volik¹,

Alcaide

Morin

et al. 2016

Molecular Cancer Research

295

235

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“…The same applies to the optimal collecting tube, where the use of anti-coagulants is favored, and it is largely agreed that the sampling procedure must be performed with great care to avoid cell lysis and contamination with DNA from normal blood cells [29]. In addition, there seems to be some consensus that a two-hour delay between sampling and blood processing is acceptable, whereas storage temperature seems less important [24,30,31]. In contrast, disagreement prevails regarding the centrifugation procedures in terms of the level of gravity (g) and regarding the use of one-or two-step procedures to ensure adequate separation and to avoid contamination and degradation prior to DNA purification and quantification procedures [24,31].…”

Section: Metastatic Colorectal Cancermentioning

confidence: 99%

“…The lack of direct comparison between studies and the numerous different quantification approaches renders progress in terms of clinical relevance difficult. Among others, the different nontumor-specific reference genes comprise cyclophilin, B2M, and albumin [26][27][28][31][32][33]; and other strategies include methylation assays or quantification of tumor-relevant genetic alterations [34]. Provided that all the above-mentioned steps are carefully considered, a reliable quantitative measure of total cfDNA is achievable and samples can be further analyzed to detect tumor-specific mutations and quantify the mutated alleles in the sample.…”

Section: Metastatic Colorectal Cancermentioning

confidence: 99%

Methodological, biological and clinical aspects of circulating free DNA in metastatic colorectal cancer

Spindler

2016

Acta Oncologica

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Background: Circulating DNA can be used to measure the total cell-free DNA (cfDNA) and for detection and quantification of tumor-specific genetic alterations in the peripheral blood, and the broad clinical potential of circulating DNA has attracted increasing focus over the past decade. Concentrations of circulating DNA are high in metastatic colorectal cancer (CRC), and the total levels of cfDNA have been reported to hold strong prognostic value. Colorectal tumors are characterized by a high frequency of well known, clinically relevant genetic alteration, which is readily detected in the cfDNA and holds potential for tailoring of palliative therapy and for monitoring during treatment. This review aims to present the current literature which has specifically reported data on the potential utility of cfDNA and on tumor-specific mutations in metastatic colorectal cancer (mCRC). Method: Methodological, biological and clinical aspects are discussed based on the most recent development in this specific setting, and eligible studies were identified by systematic literature searched from Pubmed and EMBASE in addition to conference papers and communications. Results: The literature regarding cfDNA in CRC is broad and heterogeneous concerning aims, nomenclature, methods, cohorts and clinical endpoints and consequently difficult to include in a single systematic search. However, the available data underline a strong clinical value of measuring both total cfDNA levels and tumor-specific mutations in the plasma of patients with mCRC, pre-and during systemic therapy. Conclusion: This paper had gathered the most recent literature on several aspects of cfDNA in mCRC, including methodological, biological and clinical aspects, and discussed the large clinical potential in this specific setting, which needs to be validated in carefully designed prospective studies in statistically relevant cohorts.

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“…An alternative and minimally invasive source of tumor DNA is circulating cell-free DNA that can be isolated from plasma or serum, an approach described as liquid biopsy [1]. Various applications of circulating tumor DNA analysis in clinical oncology research have been proposed with some of them now reaching routine clinical testing [2,3]. All these applications require highly sensitive procedures like digital PCR or barcoded next-generation sequencing approaches to detect the minuscule amounts of ctDNA among a majority of wild-type cfDNA [4].…”

Section: Introductionmentioning

confidence: 99%

Performance of Streck cfDNA Blood Collection Tubes for Liquid Biopsy Testing

et al. 2016

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ObjectivesMaking liquid biopsy testing widely available requires a concept to ship whole blood at ambient temperatures while retaining the integrity of the cell-free DNA (cfDNA) population and stability of blood cells to prevent dilution of circulating tumor DNA (ctDNA) with wild-type genomic DNA. The cell- and DNA-stabilizing properties of Streck Cell-Free DNA BCT blood collection tubes (cfDNA BCTs) were evaluated to determine if they can be utilized in combination with highly sensitive mutation detection technologies.MethodsVenous blood from healthy donors or patients with advanced colorectal cancer (CRC) was collected in cfDNA BCTs and standard K2EDTA tubes. Tubes were stored at different temperatures for various times before plasma preparation and DNA extraction. The isolated cfDNA was analyzed for overall DNA yield of short and long DNA fragments using qPCR as well as for mutational changes using BEAMing and Plasma Safe-Sequencing (Safe-SeqS).ResultsCollection of whole blood from healthy individuals in cfDNA BCTs and storage for up to 5 days at room temperature did not affect the DNA yield and mutation background levels (n = 60). Low-frequency mutant DNA spiked into normal blood samples as well as mutant circulating tumor DNA in blood samples from CRC patients collected in cfDNA BCTs were reliably detected after 3 days of storage at room temperature. However, blood samples stored at ≤ 10°C and at 40°C for an extended period of time showed elevated normal genomic DNA levels and an abnormally large cellular plasma interface as well as lower plasma volumes.ConclusionWhole blood shipped in cfDNA BCTs over several days can be used for downstream liquid biopsy testing using BEAMing and Safe-SeqS. Since the shipping temperature is a critical factor, special care has to be taken to maintain a defined room temperature range to obtain reliable mutation testing results.

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Cell-free DNA: Preanalytical variables

Cited by 148 publications

References 70 publications

Cell-free DNA (cfDNA): Clinical Significance and Utility in Cancer Shaped By Emerging Technologies

Cell-free DNA (cfDNA): Clinical Significance and Utility in Cancer Shaped By Emerging Technologies

Methodological, biological and clinical aspects of circulating free DNA in metastatic colorectal cancer

Performance of Streck cfDNA Blood Collection Tubes for Liquid Biopsy Testing

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