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Performance of four platforms for KRAS mutation detection in plasma cell-free DNA: ddPCR, Idylla, COBAS z480 and BEAMing.

Author(s): Vessies DCL, Greuter MJE, van Rooijen KL, Linders TC, Lanfermeijer M, Ramkisoensing KL, Meijer GA, Koopman M, Coupé VMH, Vink GR, Fijneman RJA, van den Broek D

Publication: Sci Rep, 2020, Vol. 10, Page 8122

PubMed ID: 32415199 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of the study was to compare KRAS mutation detection among four analytical methods and investigate the effects of cfDNA extraction method, MAF, and cfDNA input amount. Blood was collected from 17 patients into Streck Cell-free DNA BCT tubes and shipped within the Netherlands. Sixteen of the patients were selected based on the presence of KRAS mutations in tissue biopsies and the final patient was KRAS wildtype. After arrival, plasma was obtained by centrifugation for 10 minutes at 1,700 x g followed by 10 minutes at 20,000 x g and stored at -80°C. cfDNA was isolated from six patients using platform-specific methodology (Idylla ctKRAS Mutation Test, KRAS Mutation Test v2 LSR Kit, or OncoBEAM RAS CRC Kit RUO) and from the remaining 11 patients using the QIAsymphony Circulating DNA Kit. KRAS mutations were detected using Idylla ctKRAS Mutation Test, COBAS KRAS Mutation Test v2 LSR Kit, OncoBEAM RAS CRC Kit RUO, and BioRad ddPCR the KRAS G12/G13 Screening Kit.

   Summary of Findings:

   When cFDNA was extracted using platform-specific methodology, the KRAS mutation was detected using all four methods in two of five patients, using three of four methods in two patients (one not detected by Idylla and the other not detected by Cobas), and KRAS mutations were not detected using any of the methods in the final patient. Interestingly, a G12 mutation was identified in the cfDNA of the patient without a known KRAS mutation in the tumor specimen by Idylla and BEAMing. The KRAS results were concordant among all four methods for six of eleven patients when extraction was with QIAsymphony. Of the remaining five patients, one had the expected mutation detected by all methods except BioRad ddPCR, two had the expected mutation only identified by Idylla, the remaining two had a G12 insertion and a KRAS amplification not targeted by any of the platforms, but BEAMing identified a G12 mutation in the patient with an insertion. When a minimum of 10 ng DNA was used and only targeted mutations were considered, there was 100% concordance in KRAS mutations among the four methods. Using a synthetic reference sample, the sensitivity of the methods ranged from 10% with COBAS to 65% with BioRad ddPCR, but when only specimens with a MAF of 0.5% or higher were considered, sensitivity was 19% with COBAS to 100% with BioRad ddPCR. Using the data from the reference samples, the authors calculated that when 50 ng DNA was used, KRAS mutations at a MAF would be detected in 38% of patients by BioRad ddPCR, 32% by BEAMing, 22% by Idylla, and 17% by COBAS z480. At a MAF of 0.02% and an input volume of 50 ng, the mutation would be identified in 22% of patients by ddPCR, 11% by BEAMing, 8% by Idylla, and 0% by COBAS z480.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	PCR
   DNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	QIAsymphony Circulating DNA kit Platform-specific methodology
   Digital PCR Specific	Template/input amount	Not consistent 10 ng 50 ng
   Digital PCR Specific	Technology platform	Bio-Rad KRAS G12/G13 screening kit BioCartis Idylla Roche COBAS z480 Sysmex BEAMing

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