Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Performance comparison of commercial kits for isolating and detecting of circulating tumor DNA.

Author(s): Wang M, Huang X, Li X, Guo Q, Xu W, Zhao M, Wang X, Wang L, Lou J

Publication: Scand J Clin Lab Invest, 2021, Vol. , Page 1-6

PubMed ID: 33999736 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared DNA yield, percentage of 100-300 bp DNA fragments, amplificability, and recovery of different size DNA fragments when DNA was extracted from pooled plasma using four different kits. The ability to detect EGFR mutations in cfDNA was also compared between two methods. EDTA blood was collected from 56 healthy patients, 14 patients with early stage lung cancer, and 21 patients with advanced stage lung cancer. Plasma was obtained by centrifugation at 1600 x g for 10 min at 4°C within 2 h of collection. The plasma was then centrifuged at 16,000 x g for 10 min at 4°C and specimens were pooled based on diagnosis to create four plasma pools consisting of: plasma from 22 healthy patients, plasma from the patients with early stage lung cancer, plasma from patients with late stage lung cancer, or plasma from the remaining 34 healthy patients mixed with 71 bp, 173 bp, and 3988 bp fragments of plasmids. Plasma pools were stored at -80°C. DNA was isolated in triplicate from plasma pools using the QIAamp Circulating Nucleic Acid Kit, AmoyDx Circulating DNA Kits, Microdiag Circulating DNA Isolation Kit, or MagMAX Cell-free DNA Isolation Kit. DNA was quantified using Qubit 3.0 and the size profile evaluated using an Agilent 2100 Bioanalyzer. To investigate potential PCR inhibition, EGFR and b-Actin were amplified by real-time PCR. EGFR mutations were detected using the MicroDiag EGFR Gene Mutation Detection Kit and the Amoy Diagnostics Real-time PCR Detection Kit for the Analysis of EGFR Gene Mutations.

   Summary of Findings:

   The DNA yield was highest when extraction was with the QIAamp Circulating Nucleic Acid Kit, followed by the Microdiag Circulating DNA Isolation Kit (MB), and the MagMAX Cell-free DNA Isolation Kit with the lowest yields obtained using the AmoyDx Circulating DNA Kits. Significance of the differences depended on the diagnostic pool used. The absolute yield and percentage of 100-300 bp DNA fragments was lowest using the AmoyDx Circulating DNA Kits for all three pools. Although the highest yield of 100-300 bp DNA in the pool of plasma from healthy patients and the pool from patients with advanced lung cancer was found when extraction was with the MagMAX cell-free DNA Isolation Kit, the percentage of 100-300 bp DNA fragments in these same pools was highest when extraction was with the Microdiag Circulating DNA Isolation Kit. In contrast, the highest yield of 100-300 bp DNA was found in the pooled plasma from patients with early stage lung cancer when extraction was with the Microdiag Circulating DNA Isolation Kit, but the greatest percentage of DNA was 100-300 bp when extraction was with MagMAX cell-free DNA Isolation Kit. Recovery of spiked-in DNA showed that for the QIAamp Circulating Nucleic Acid Kit, AmoyDx Circulating DNA Kits, and MagMAX Cell-free DNA Isolation Kit, the efficiency increased with fragment size. The QIAamp Circulating Nucleic Acid Kit had the best recovery of 173 bp fragments. PCR amplification of EGFR and B-Actin were highest from the healthy plasma pool and the pooled plasma from the early lung cancer patients when extraction was with the QIAamp Kit, but the amplification was highest from the advanced lung cancer pool when extraction was with MagMAX cell-free DNA Isolation Kit, although the authors report PCR inhibition on QIAamp extracts. Both assays successfully detected all four EGFR mutations at 1% MF and L858R, G719S, and 19-Del mutations at 0.5% MF with T790M mutation at 0.5% MF having a sensitivity of 87.5% using MicroDiag EGFR Gene Mutation Detection Kit but only 37.5% using the fluorometric real-time PCR detection kit. Detection of mutations with a frequency of 0.1% was more variable with 100% of L858R and 40% of 19-Del but 0% of T790M and G719S were detected using the fluorometric real-time PCR detection kit and 25% of 19-Del and T790M and 0% of the L858R and G719S mutations detected using the MicroDiag EGFR Gene Mutation Detection Kit.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   DNA	Fluorometry
   DNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Early stage lung cancer Advanced stage lung cancer Healthy
   Real-time qPCR Specific	Targeted nucleic acid	EGFR G719S EGFR T790M EGFR 19-Del EGFR L858R
   Real-time qPCR Specific	Technology platform	MicroDiag EGFR Gene Mutation Detection Kit Amoy Diagnostics fluorometric real-time PCR detection kit for the analysis of EGFR gene mutations
   Analyte Extraction and Purification	Analyte isolation method	AmoyDx Circulating DNA Kits QIAamp Circulating Nucleic Acid Kit Microdiag Circulating DNA Isolation Kit MagMAX Cell-free DNA Isolation Kit

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