Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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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

Conclusion of Paper

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

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	Automated electrophoresis/Bioanalyzer
   DNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(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

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