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Validation of a next-generation sequencing assay for the detection of EGFR mutations in cell-free circulating tumor DNA.

Author(s): Stitz R, Buder A, Silye R, Baumgartner B, Pühringer F, Filipits M, Oberndorfer E, Heitzer E

Publication: Exp Mol Pathol, 2021, Vol. 123, Page 104685

PubMed ID: 34560086 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effect of cfDNA input amount and VAF on detection of EGFR mutations in plasma by NGS. The sensitivity of the NGS assay was determined by comparing NGS results to results of ddPCR. Blood from 16 patients with non-small cell lung cancer (NSCLC) was collected into two or three cell-free DNA BCT. Plasma was separated by centrifugation at 300 x g for 10min followed by 5000 x g for 10 min. cfDNA was isolated using the QIAmp Circulating Nucleic Acid Kit on a QIAcube and quantified using the Qubit dsDNA HS Assay Kit. DNA was analyzed fresh or stored frozen at -20°C. The EGFR activating mutations, L858R and exon 19 deletion, and the EGFR resistance mutation T790M were detected and quantified using the QX-100 ddPCR system and the next generation sequencing-based Archer Reveal ctDNA assay.

   Summary of Findings:

   In the NGS assay, DNA input (14-181 ng DNA) was strongly correlated with coverage (R2=0.7283) but not read count. Coverage for the EGFR variant T790M was low with only 27% of molecules sequenced at that position. The allele frequency necessary to distinguish a variant from background in 50% of cases (ND MDAF) ranged between 0.08 and 0.29% for the hotspot positions when 22 ng DNA was used and between 0.03 and 0.20% when 57 ng DNA was used. EGFR T790M and NRAS G12C had higher noise than other positions.

   Of the 16 patient tumors evaluated, six carried the L858R mutation and 10 had exon 19 deletions. Mutations were detected by both assays (ddPCR and NGS) in plasma from eight patients, only by ddPCR in plasma from one patient, and only by NGS in plasma from two patients; no mutations were detected in plasma from the remaining five patients. Detection of the L858R mutation was 100% concordant between NGS and ddPCR assays. Detection of the Exon 19 deletion was concordant in five patients; in one patient the Exon 19 deletion was detected by ddPCR at low copy number in one of three specimens, in another patient the mutation was detected by NGS at very low copy number in just one of three specimens, in another patient the mutation was detected by NGS at a copy number below the limit of detection and by ddPCR in one patient and was not detected in the remaining three patients. The T790M mutation was detected by both NGS and ddPCR in two patients, at very low copy number by only ddPCR in two patients and was not detected in the remaining 11 patients.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Next generation sequencing
   DNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Next generation sequencing Specific	Template/input amount	14-181 ng DNA
   Digital PCR Specific	Targeted nucleic acid	EGFR L858R EGFR T790M EGFR Exon 19 del
   Next generation sequencing Specific	Targeted nucleic acid	EGFR T790M EGFR Exon 19 del EGFR L858R
   Next generation sequencing Specific	Technology platform	ddPCR

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