Influence of Plasma Processing on Recovery and Analysis of Circulating Nucleic Acids

Page, Karen M.; Guttery, David S.; Zahra, Nathalie; Primrose, Lindsay; Elshaw, Shona R.; Pringle, J. H.; Blighe, Kevin; Marchese, S; Hills, Alice; Woodley, Laura; Stebbing, Justin; Coombes, R. Charles; Shaw, Jacqueline A

doi:10.1371/journal.pone.0077963

Cited by 171 publications

(154 citation statements)

References 29 publications

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“…Plasma is theoretically less likely to be contaminated with DNA from blood cells but, importantly, the time elapsed between blood collection and centrifugation can heavily influence this (41). Unfortunately, because the overall yield of DNA extracted from plasma or the amount of DNA measured by qPCR is often used as a proxy for cfDNA, there is also conflicting evidence on the extent of contamination from the blood (42). As revisited later, contamination may be best assessed by determining the relative abundance of high molecular weight DNA fragments that are not consistent with the apoptotic signature characteristic of cfDNA.…”

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

302

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

302

235

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“…These were unselected, sequential patients attending the Breast Clinic at Charing Cross Hospital. Blood was collected into EDTA-containing tubes (BD Biosciences) and processed to obtain plasma as described previously (20 ). For CTCs, 7.5 mL of blood was collected into CellSave TM collection tubes (Veridex LLC; Raritan).…”

Section: Blood Processing For Plasma and Ctc Enumerationmentioning

confidence: 99%

Noninvasive Detection of Activating Estrogen Receptor 1 (ESR1) Mutations in Estrogen Receptor–Positive Metastatic Breast Cancer

et al. 2015

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BACKGROUND Activating mutations in the estrogen receptor 1 (ESR1) gene are acquired on treatment and can drive resistance to endocrine therapy. Because of the spatial and temporal limitations of needle core biopsies, our goal was to develop a highly sensitive, less invasive method of detecting activating ESR1 mutations via circulating cell-free DNA (cfDNA) and tumor cells as a “liquid biopsy.” METHODS We developed a targeted 23-amplicon next-generation sequencing (NGS) panel for detection of hot-spot mutations in ESR1, phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), tumor protein p53 (TP53), fibroblast growth factor receptor 1 (FGFR1), and fibroblast growth factor receptor 2 (FGFR2) in 48 patients with estrogen receptor-α–positive metastatic breast cancer who were receiving systemic therapy. Selected mutations were validated using droplet digital PCR (ddPCR). RESULTS Nine baseline cfDNA samples had an ESR1 mutation. NGS detected 3 activating mutations in ESR1, and 3 hot-spot mutations in PIK3CA, and 3 in TP53 in baseline cfDNA, and the ESR1 p.D538G mutation in 1 matched circulating tumor cell sample. ddPCR analysis was more sensitive than NGS and identified 6 additional baseline cfDNA samples with the ESR1 p.D538G mutation at a frequency of <1%. In serial blood samples from 11 patients, 4 showed changes in cfDNA, 2 with emergence of a mutation in ESR1. We also detected a low frequency ESR1 mutation (1.3%) in cfDNA of 1 primary patient who was thought to have metastatic disease but was clear by scans. CONCLUSIONS Early identification of ESR1 mutations by liquid biopsy might allow for cessation of ineffective endocrine therapies and switching to other treatments, without the need for tissue biopsy and before the emergence of metastatic disease.

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“…All patients gave written informed consent prior to participation. Blood was taken by venepuncture into K2-EDTA-containing collection tubes (BD Biosciences, San Jose, CA, USA) and processed for cfDNA isolation as described in Page and colleagues [15].…”

Section: Dna Reference Samplementioning

confidence: 99%

Peptide nucleic acid clamping to improve the sensitivity of Ion Torrent-based detection of an oncogenic mutation in <em>KRAS</em>

Rakhit

Ottolini

Jones

et al. 2017

Matters

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Detecting oncogenic changes in the genome of cancer patients is crucial for targeted therapy. Such changes include alterations to KRAS, a GTPase located upstream of several signalling transduction pathways implicated in cancer formation. While nextgeneration sequencing (NGS) allows for comprehensive analysis of a genome, the technology can struggle to detect low frequency variants. To improve the sensitivity of NGS for detecting mutations we created a peptide nucleic acid (PNA) clamp, validated by qPCR, designed to bind wild-type KRAS (WT KRAS) across codon 12 during the PCR amplification stage of a NGS library preparation. We tested the effect of clamping the wild-type KRAS sequence in a reference standard with a KRAS c.35G>A mutation (KRAS G12D ) at an allelic frequency (AF) of 1.3% and on circulating-free DNA from a patient harbouring a KRAS G12D mutation (at an AF of 3.2%). Runs were conducted using 10, 5, 2.5 and 1 ng of DNA input. The PNA increased the number of mutant reads and their frequency relative to wild-type calls, allowing for more sensitive detection at all tested concentrations of DNA input.

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Influence of Plasma Processing on Recovery and Analysis of Circulating Nucleic Acids

Cited by 171 publications

References 29 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

Noninvasive Detection of Activating Estrogen Receptor 1 (ESR1) Mutations in Estrogen Receptor–Positive Metastatic Breast Cancer

Peptide nucleic acid clamping to improve the sensitivity of Ion Torrent-based detection of an oncogenic mutation in <em>KRAS</em>

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