NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

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

Author(s): Pérez-Barrios C, Nieto-Alcolado I, Torrente M, Jiménez-Sánchez C, Calvo V, Gutierrez-Sanz L, Palka M, Donoso-Navarro E, Provencio M, Romero A

Publication: Transl Lung Cancer Res, 2016, Vol. 5, Page 665-672

PubMed ID: 28149760 PubMed Review Paper? No

Purpose of Paper

This paper compared three extraction kits by measuring cell-free DNA (cfDNA) concentration, fragment size, and mutant allele frequency (MAF) in plasma of cancer patients. The effects of cancer stage and tumor location on cfDNA concentration were also investigated in some specimens.

Conclusion of Paper

The median cfDNA concentrations were comparable between specimens extracted with the Maxwell RSC Kit and QIAamp CNA Kit but were lower when extracted using the MagNA Pure Kit than with the Maxwll RSC Kit. The concentration of cfDNA was unaffected by tumor location but was higher in patients with stage IV cancer than those without metastatic disease. While all extraction methods yielded cfDNA fragments of 151-201 bp, 69 of 78 cfDNA extractions also had mono-, di-, and tri-nucleosome peaks and 54 specimens had high molecular weight DNA. Compared to the Maxwell RSC Kit, extraction with the MagNa Pure Kit resulted in a higher proportion of cfDNA fragments between 150 and 200 bp, lower proportion of cfDNA bound to di- or tri-nucleosomes, and comparable MAF. A comparable MAF was also observed for specimens extracted using the QIAamp CNA and Maxwell RSC Kits.

Studies

  1. Study Purpose

    This study compared extraction kitsby measuring cfDNA concentration, fragment size, and MAF in plasma of cancer patients. The effects of cancer stage and tumor location on cfDNA concentration were also investigated in some specimens. Blood from 47 patients with lung cancer and 10 patients with colon cancer was collected into PPT tubes with a gel barrier. Plasma was obtained by centrifugation at 1500 x g for 10 min at 4˚C within 2 h of collection. Plasma was aliquoted and stored at -80˚C. Plasma was thawed in the refrigerator and centrifuged at 5000 x g for 20 min before cfDNA extraction. cfDNA was extracted from 31 specimens (30 lung cancer and 1 colon cancer) using the Maxwell RSC ccfDNA Plasma Kit and QIAamp Circulating Nucleic Acid (CNA) kit, from 24 specimens using the MagNA Pure Compact Nucleic Acid Isolation Kit I and the Maxwell RSC ccfDNA Plasma Kit, and from the remaining two specimens using all three methods. cfDNA concentration was determined by Qubit dsDNA HS Assay and fragment length was investigated by bioanalyzer. cfDNA mutations were quantified by digital PCR amplification of EGFR p.T790M, p.L858R, and p.E746_A750delELREA and KRAS p.G12V, p.G12D,  and p.G13D.

    Summary of Findings:

    The median cfDNA concentrations were comparable between specimens extracted with the Maxwell RSC Kit and QIAamp CNA Kit (1.25 ng/µL versus 1.08 ng/µL, P=0.775), but were lower when extracted using the MagNA Pure Kit than with the Maxwll RSC Kit (1.154 ng/µL versus 2.31 ng/µL, P<0.0001). The concentration of cfDNA was higher in patients with stage IV cancer than those without metastatic disease, regardless of extraction method (P=0.045 for Maxwell RSC and P=0.0173 for QIAamp CNA), but the cfDNA concentration was not affected by tumor localization, regardless of whether extraction was with the Maxwell RSC Kit or the MagNa Pure Kit.

    All extraction methods yielded cfDNA fragments of 151-201 bp, but 69 of 78 cfDNA extractions also had mono-, di-, and tri-nucleosome peaks and 54 specimens had high molecular weight DNA. Importantly, the proportion of cfDNA fragments between 150 and 200 bp was significantly higher when extracted using the MagNa Pure Kit than the Maxwell RSC Kit (P=0.0005). Conversely, extraction using the Maxwell RSC Kit rather than the MagNa Pure Kit resulted in a higher proportion of cfDNA bound to dinucleosomes (P=0.0024) and trinucleosomes (P=0.038).

    For 19 of 26 patients, KRAS mutations found in FFPE specimens were confirmed in cfDNA. Importantly, the mutant allele frequency did not differ between cfDNA obtained using the MagNa Pure and Maxwell RSC kits or the QIAamp CNA and Maxwell RSC kits.

    Biospecimens
    Preservative Types
    • Frozen
    Diagnoses:
    • Neoplastic - Carcinoma
    Platform:
    AnalyteTechnology Platform
    DNA Fluorometry
    DNA Bioanalyzer
    DNA Digital PCR
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Preaquisition Diagnosis/ patient condition Lung cancer
    Colon cancer
    Stage III cancer
    Stage IV cancer
    Analyte Extraction and Purification Analyte isolation method MagNA Pure Compact Nucleic Acid Isolation Kit I versus Maxwell RSC ccfDNA Plasma Kit
    QIAamp Circulating Nucleic Acid kit versus Maxwell RSC ccfDNA Plasma Kit

You Recently Viewed  

comments powered by Disqus

News and Announcements

  • Most Popular SOPs in 2018

  • New Article Published from NCI's Biospecimen Pre-analytical Variables Program

  • SOPs from the Biospecimen Pre-analytical Variables (BPV) Program are now available!

  • More...