Comparison of methods for circulating cell-free DNA isolation using blood from cancer patients: impact on biomarker testing

Pérez-Barrios, Clara; Nieto-Alcolado, I.; Torrente, María; Sánchez, Carolina Jiménez; Calvo, Virginia; Gutiérrez-Sanz, Lourdes; Palka, Magda; Donoso-Navarro, Encarnación; Provencio, Mariano; Romero, Atocha

doi:10.21037/tlcr.2016.12.03

Cited by 119 publications

(87 citation statements)

References 20 publications

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Section: Cfdna Extraction From Blood and Other Materialsmentioning

confidence: 99%

“…Since interaction effects between the method for cfDNA extraction and the method applied for mutation detection may lead to increased artifacts, careful comparison during establishing liquid biopsy procedures is warranted. 78,83 Different abilities to recover longer or shorter fragments have been described in the literature. 84,85 Besides biological and pathological parameters reviewed above in chapter 1, the concentration of isolated cfDNA is influenced by the efficiency of the DNA extraction method and the final elution volume of the respective method.…”

Section: Cf D Na E Xtr Ac Ti On Fr Om Bl Ood a Nd Oth E R M A Te Rimentioning

confidence: 99%

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A field guide for cancer diagnostics using cell‐free DNA: From principles to practice and clinical applications

Volckmar

Sültmann

Riediger

et al. 2017

Genes Chromosomes & Cancer

169

151

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Recently, many genome-wide profiling studies provided insights into the molecular make-up of major cancer types. The deeper understanding of these genetic alterations and their functional consequences led to the discovery of novel therapeutic opportunities improving clinical management of cancer patients. While tissue-based molecular patient stratification is the gold standard for precision medicine, it has certain limitations: Tissue biopsies are invasive sampling procedures carrying the risk of complications and may not represent the entire tumor due to underlying genetic heterogeneity. In this context, complementary characterization of genetic information in the blood of cancer patients can serve as minimal-invasive 'liquid biopsy'. Fragments of circulating cell-free DNA (cfDNA) are released from tissues of healthy individuals as well as cancer patients. The fraction of cfDNA that is released from primary tumors or metastases (i.e. circulating tumor DNA, ctDNA) represents genetic aberrations in cancer cells, which are a potential source for diagnostic, prognostic, and predictive biomarkers. Recent studies have demonstrated technical feasibility and clinical applications including detection of drug targets and resistance mutations as well as longitudinal monitoring of tumors under therapy. To this end, a variety of pre-analytical procedures for blood processing, isolation and quantification of cfDNA are being employed and several analytical methods and technologies ranging from PCR-based single locus assays to genome-wide approaches are available, which considerably differ in sensitivity, specificity, and throughput. However, broad implementation of ctDNA analysis in daily clinical practice requires a thorough understanding of theoretical, technical, and biological concepts and necessitates standardization and validation of pre-analytical and analytical procedures across different technologies. Here, we review the pertinent literature and discuss the advantages and limitations of available methodologies and their potential applications in molecular diagnostics.

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Section: Cfdna Extraction From Blood and Other Materialsmentioning

confidence: 99%

Section: Cf D Na E Xtr Ac Ti On Fr Om Bl Ood a Nd Oth E R M A Te Rimentioning

confidence: 99%

A field guide for cancer diagnostics using cell‐free DNA: From principles to practice and clinical applications

Volckmar

Sültmann

Riediger

et al. 2017

Genes Chromosomes & Cancer

169

151

View full text Add to dashboard Cite

show abstract

“…When tested using identical platforms, some studies report good agreement in the mutation status between cfDNA and FFPE [44,70]. Higgins et al, found 100% concordance of PIK3CA mutation status by BEAMing between FFPE samples and corresponding cfDNA from blood [71].…”

Section: Methods Targeting Druggable Mutation and Other Aberrations Imentioning

confidence: 99%

“…Similarly, Grasselli and colleagues also saw very high heterogeneity and poor correlation in the mutational load between tissueplasma results [75]. These differences could potentially explained by low tumor burden [75] or ctDNA shedding or low total number of tumor cells in the primary tumor [76] or due to differences in protocol for isolating DNA from FFPE and plasma [44]. Also, formalin fixation introduces DNA denaturation, and introduction of nonreproducible sequence alterations in DNA [77].…”

Section: Methods Targeting Druggable Mutation and Other Aberrations Imentioning

confidence: 99%

“…Most authors use QIAamp Circulating Nucleic Acid (Qiagen) which utilizes magnetic bead based technology to isolate plasma tumor derived DNA. A comparison of three popular kits by authors Pérez-Barrios and group found no significant differences in recovery of cfDNA extracted using kit from Qiagen or Maxwell® RSC ccfDNA Plasma Kit (Promega) but the cfDNA yield from MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche) was significantly less [44]. But others have found that the yield of cfDNA using the traditional phenol/chloroform/ethanol method in the presence of glycogen was better than that isolated in the absence of glycogen or by commercially available kit [45].…”

Section: Extraction Of Molecular Informationmentioning

confidence: 96%

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Liquid biopsy - emergence of a new era in personalized cancer care

2018

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The most successful treatment for cancer involves identifying druggable, biological markers for targeted therapy. In the clinical setting, surgical removal of tumors is the only procedure for identifying such targetable molecules. Shed from tumor cells, these markers are also present in circulating blood, albeit in very negligible amounts. Liquid biopsy is a procedure performed on a blood sample to look for such circulating cancer markers cells or pieces of nucleic acid from the tumor. The procedure shows promise in revolutionizing personalized cancer treatments. Here we briefly review the technique, characterization, and its utilization in clinics.

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High‐throughput isolation of circulating tumor DNA: a comparison of automated platforms

et al. 2018

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The emerging interest in circulating tumor DNA (ctDNA) analyses for clinical trials has necessitated the development of a high‐throughput method for fast, reproducible, and efficient isolation of ctDNA. Currently, the majority of ctDNA studies use the manual QIAamp (QA) platform to isolate DNA from blood. The purpose of this study was to compare two competing automated DNA isolation platforms [Maxwell (MX) and QIAsymphony (QS)] to the current ‘gold standard’ QA to facilitate high‐throughput processing of samples in prospective trials. We obtained blood samples from healthy blood donors and metastatic cancer patients for plasma isolation. Total cell‐free DNA (cfDNA) quantity was assessed by TERT quantitative PCR. Recovery efficiency was investigated by quantitative PCR analysis of spiked‐in synthetic plant DNA. In addition, a β‐actin fragmentation assay was performed to determine the amount of contamination by genomic DNA from lysed leukocytes. ctDNA quality was assessed by digital PCR for somatic variant detection. cfDNA quantity and recovery efficiency were lowest using the MX platform, whereas QA and QS showed a comparable performance. All platforms preferentially isolated small (136 bp) DNA fragments over large (420 and 2000 bp) DNA fragments. Detection of the number variant and wild‐type molecules was most comparable between QA and QS. However, there was no significant difference in variant allele frequency comparing QS and MX to QA. In summary, we show that the QS platform has comparable performance to QA, the ‘gold standard’, and outperformed the MX platform depending on the readout used. We conclude that the QS can replace the more laborious QA platform, especially when high‐throughput cfDNA isolation is needed.

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Comparison of methods for circulating cell-free DNA isolation using blood from cancer patients: impact on biomarker testing

Abstract: Methods for isolation of cfDNA can affect DNA yield and molecular weight fractions recovery. These observations should be taken into account for cfDNA analysis in routine clinical practice.

Cited by 119 publications

References 20 publications

A field guide for cancer diagnostics using cell‐free DNA: From principles to practice and clinical applications

A field guide for cancer diagnostics using cell‐free DNA: From principles to practice and clinical applications

Liquid biopsy - emergence of a new era in personalized cancer care

High‐throughput isolation of circulating tumor DNA: a comparison of automated platforms

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