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Performance characteristics of a polymerase chain reaction-based assay for the detection of EGFR mutations in plasma cell-free DNA from patients with non-small cell lung cancer using cell-free DNA collection tubes.

Author(s): May T, Clement MS, Halait H, Kohlmann A, Kohlmann M, Lai J, Lee N, Li-Sucholeiki X, Meldgaard P, Joshi S, Scudder S, Shrestha N, Sorensen B, Kiral M, O'Donnell P

Publication: PLoS One, 2024, Vol. 19, Page e0295987

PubMed ID: 38593164 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared EGFR mutation detection between specimens collected in Roche Cell-Free DNA (cfDNA) Collection Tubes and K2EDTA tubes, among plasma from blood specimens collected in cfDNA tubes that were inverted 2-58 times, and among plasma obtained from cfDNA tubes using different centrifugation protocols. The stability of specimens collected in cfDNA tubes was also investigated by comparing EGFR mutation detection after whole blood was stored for 25 hours at 32˚C followed by storage at 27˚C for ≤8 days or after plasma was stored at 32˚C for ≤25 hours, 2-8˚C for ≤8 days, or at -25˚C to -15˚C or -70˚C for ≤13 months with three freeze-thaw cycles. The reproducibility of the assay and the effects of contamination with high levels of hemoglobin, triglycerides, bilirubin, albumin, or the preservative solution were also investigated. To compare tube types, blood was collected from 17 patients with mutation-positive NSCLC, 34 patients with mutation-negative NSCLC, and 20 healthy patients (spiked with EGFR mutations) into Roche Cell-Free DNA (cfDNA) Collection Tubes and K2EDTA tubes. To investigate specimen stability, blood from patients (unspecified number) with NSCLC was stored for 25 h at 32˚C followed by storage at 27˚C for 4, 7, or 8 days and plasma was stored at 32˚C for 5 or 25 hours, at 2-8˚C for 4 or 8 days, or at -25˚C to -15˚C or –70˚C for 31 days or 13 months with 3 freeze-thaw cycles (details not provided).  Potential effects of tube inversions were investigated by inverting tubes of whole blood collected from healthy volunteers for 0, 2, 4, 6, 8, or 28 times, then adding sheared DNA from a cell line that contains EGFR mutations, and inverting the tubes 2, 4, 6, 8, 10, or 30 times, respectively; plasma was subsequently separated. The effect of centrifugation speed was investigated by mixing whole blood from healthy volunteers DNA from a cell line that contains EGFR mutations; separating  plasma by centrifugation at 2600 g for 9 or 16 min, at 2600 g for 12.5 min, or at 1100 g for 9 or 16 min; and analysis with a Cobas EGFR system. The limit of detection was verified using plasma from patients with NSCLC that was spiked at the limit of detection (75 or 100 copies/mL) with EGFR mutations and was then validated by creating two spiked specimens and analyzing them separately. Assay reproducibility was tested by spiking blood from healthy donors into three different lots of Roche cfDNA tubes and analyzing samples at three different sites with two different operators on three non-consecutive days. To test for effects of interfering substances, hemoglobin (2 g/L), triglycerides (33 g/L), albumin (60 g/L), bilirubin (0.2 g/L), or Roche cfDNA preservative solution (4×) were added to plasma from healthy volunteers (spiked with EGFR mutations).  For analysis, EGFR mutations were profiled by the real-time PCR-based Cobas EGFR test and were validated in K2EDTA plasma specimens by NGS using an Illumina MiSeq System.

   Summary of Findings:

   EGFR mutation status was 100% concordant between tube types for specimens from patients with mutation-positive NSCLC, patients with mutation-negative NSCLC, and healthy patients (spiked with EGFR mutations).  EGFR mutation status was unaffected by any of the tested conditions (storage of whole blood for 25 hours at 32˚C followed by storage at 27˚C for ≤8 days; storage of plasma at 32˚C for ≤25 hours, 2-8˚C for ≤8 days, or at -25˚C to -15˚C or -70˚C for ≤13 months with 3 freeze-thaw cycles) (details not provided). While at least 4 inversions of the Roche cfDNA tubes were necessary to prevent hemolysis, the number of tube inversions did not affect the detection of mutations in plasma from blood that was spiked with DNA from a cell line that contains EGFR mutations. EGFR mutations were detected in plasma from the spiked blood regardless of the centrifugation protocol used to separate the plasma. The previously identified limit of detection for K2EDTA plasma was verified using plasma isolated from blood collected in Roche cfDNA tubes, and in paired spiked specimens that were diluted to the limit of detection and analyzed separately; the agreement in EGFR status between specimens was ≥95.8%. When spiked blood from healthy patients was placed in three different lots of Roche cfDNA tubes and analyzed at three different sites with two different operators on three non-consecutive days the agreement between specimens was ≥98.6%, with a coefficient of variation for cycle threshold value of 4.4-13.7% across all mutations. There was no effect of high levels of hemoglobin, triglycerides, bilirubin, albumin, or the preservative solution on the detection of EGFR mutations using the Cobas EGFR test.

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • Frozen
   • Streck/BCT
   • None (Fresh)

   Diagnoses:

   • Normal
   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Diagnosis/ patient condition	Mutation positive NSCLC Mutation negative NSCLC Healthy
   Biospecimen Aliquots and Components	Biospecimen mixing	Inverted 0 times then twice Inverted 2 times then 4 times Inverted 4 times then 6 times Inverted 6 times then 8 times Inverted 8 times then 10 times Inverted 28 times then 30 times
   Biospecimen Acquisition	Type of collection container/solution	Roche Cell-Free DNA (cfDNA) Collection Tube K2EDTA tube
   Storage	Storage temperature	Blood at 32˚C for 25 h followed by storage at 27˚C for 4 days Blood at 32˚C for 25 h followed by storage at 27˚C for 7 days Blood at 32˚C for 25 h followed by storage at 27˚C for 8 days Plasma at 32˚C for 5 h Plasma at 32˚C for 25 h Plasma at 2-8˚C for 4 days Plasma at 2-8˚C for 8 days Plasma at –25˚C to –15˚C for 31 days with 3 freeze -thaw cycles Plasma at –25˚C to –15˚C for 13 months with 3 freeze -thaw cycles Plasma at –70˚C for 31 days with 3 freeze -thaw cycles Plasma at –70˚C for 13 months with 3 freeze -thaw cycles
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated Multiple durations compared Multiple speeds compared
   Biospecimen Aliquots and Components	Biospecimen components	Added hemoglobin (2 g/L) Added triglycerides (33 g/L) Added albumin (60 g/L) Added bilirubin (0.2 g/L) 4 times Roche cfDNA preservative solution
   Storage	Storage duration	Blood for 25 h at 32˚C followed by storage at 27˚C for 4 day Blood for 25 h at 32˚C followed by storage at 27˚C for 7 day Blood for 25 h at 32˚C followed by storage at 27˚C for 8 days Plasma for 5 h at 32˚C Plasma for 25 h at 32˚C Plasma for 4 days at 2-8˚C Plasma for 8 days at 2-8˚C Plasma for 31 days at –25˚C to –15˚C with 3 freeze -thaw cycles Plasma for 13 months at –25˚C to –15˚C with 3 freeze -thaw cycles Plasma for 31 days at –70˚C with 3 freeze -thaw cycles Plasma for 13 months at –70˚C with 3 freeze -thaw cycles
   Storage	Freeze/thaw cycling	0 cycles 3 cycles

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