Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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A novel method for liquid-phase extraction of cell-free DNA for detection of circulating tumor DNA.

Author(s): Janku F, Huang HJ, Pereira DY, Kobayashi M, Chiu CH, Call SG, Woodbury KT, Chao F, Marshak DR, Chiu RYT

Publication: Sci Rep, 2021, Vol. 11, Page 19653

PubMed ID: 34608196 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This paper investigated whether DNA yield and mutation detection were affected by cell-free DNA isolation with the QIAamp CNA, Phasify MAX and/or Phasify Enrich kits using plasma specimens from cancer patients. K₂EDTA blood was collected at multiple timepoints from patients that had a BRAF, KRAS or NRAS mutation previously identified in a tumor specimen. Plasma was obtained by centrifugation at 1900 g for 10 min at 4°C followed by 16000 g­ for 10 min at 4°C and stored at -80°C. cfDNA was isolated from plasma using QIAamp CNA, Phasify MAX and/or Phasify Enrich kits. cfDNA concentration was quantified using the Quan-iT PicoGreen dsDNA Assay Kit and fragment size was evaluated by Bioanalyzer. cfDNA was quantified by the Quant-iT PicoGreen dsDNA Assay Kit and ddPCR amplification of wildtype and mutant cfDNA. In the initial comparison of DNA extraction methods, 4 ng or 10 ng of 145 bp DNA or DNA ladder were spiked into healthy human plasma (details not provided). Potential effects of extraction on clinical specimens were investigated using 91 plasma specimens from 34 cancer patients with BRAF, KRAS or NRAS mutations present in a tumor specimen.  The authors also investigated if mutations could be detected in any of the 47 plasma specimens (from 31 cancer patients) that produced negative results after cfDNA was isolated with the QIAamp kit when extraction was with the Phasify Enrich kit.

    

   Summary of Findings:

   A higher number of copies of spiked-in DNA were recovered from plasma when the Phasify MAX kit was used compared to when the QIAamp CNA kit was used (2.1 x 10¹⁰ versus 1.1 x 10¹⁰ when spiked with 4 ng/mL; 6.4 x 10¹⁰ versus 3.5 x 10¹⁰ when spiked with 10 ng/mL). When plasma was spiked with a DNA ladder (50-300 bp), fragments of each size were successfully isolated with both QIAamp and Phasify kits but the peak heights were higher when the Phasify MAX kit was used.  Recovery of spiked-in 145 bp DNA was comparable after isolation with the Phasify Enrich kit, which incorporates an extra step to remove high genomic weight DNA, and the Phasify MAX kit, although the former method did not recover DNA >500 bp from plasma spiked with a DNA ladder (50-10,000 bp). A median of 16 ng and 10 ng of DNA were obtained from clinical plasma specimens when Phasify MAX and QIAamp kits were used for isolation, respectively. The sensitivity of the isolated cfDNA for detecting mutations identified in tissue was higher when the Phasify MAX kit was used compared to the QIAamp kit (55% versus 45%), but the specificity was 100% for both kits. For cases where the tissue mutation was detected in DNA isolated from plasma with both the PhasifyMAX kit and QIAamp kit, the average and median number of copies were 42% and 71% higher when extracted with PhasifyMAX than QIAamp (P = 0.0006),. When the Phasify Enrich and the QIAamp kit were compared DNA yield was non-significantly lower and sensitivity for detecting tissue mutations was higher when the Phasify Enrich kit was used for DNA isolation (11 ng versus 17 ng, P=0.28 and 63% versus 59%). Importantly, the number of mutation copies was higher when DNA extraction was with the Phasify Enrich kit rather than QIAamp kit (P=0.01).  In a separate cohort, the mutation present in tissue was not detectable in 47 plasma specimens processed with the QIAamp kit although the mutation was detected in 7 of those specimens when processed with the Phasify Enrich kit.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Normal
   • Neoplastic - Not specified

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Phasify MAX Kit Phasify ENRICH Kit QIAamp Circulating Nucleic Acid Kit
   Biospecimen Aliquots and Components	Aliquot size/volume	4 ng/mL DNA 10 ng/mL DNA

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