Purpose: Quantifying changes in circulating tumor DNA (ctDNA) during treatment can rapidly identify patients responding to therapy, but accurate interpretation requires an understanding of variability in ctDNA levels in the absence of treatment. This analysis of 360 patients with advanced epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC) characterized ctDNA level changes in paired pretreatment samples and evaluated factors associated with variability.
Methods: Screening and cycle 1, day 1 (C1D1) pretreatment samples from 132 previously untreated and 228 previously treated patients in the FLAURA and AURA3 trials, respectively, had ctDNA quantified using next-generation sequencing (Guardant360/GuardantOMNI NGS panel; mean variant allele frequency [VAF] or EGFR-mutant VAF) and droplet digital polymerase chain reaction (ddPCR, Biodesix) EGFR assays.
Results: In FLAURA and AURA3, ≥20% reductions in ctDNA levels (panel mean VAF) from screening to C1D1 occurred in 31/132 (23.5%) and 43/228 (18.9%) patients with paired pretreatment plasma samples, respectively, including ≥50% reductions in 12 (9.1%) and 23 (10.1%) patients, and 100% reductions (ctDNA clearance) in 0 and 5 (2.2%) patients. Background variability was not fully accounted for by intra-NGS or NGS-ddPCR technical variability or other potential sources of variability; larger changes were associated with low VAF and low cell-free DNA (cfDNA) input.
Conclusion: Intrapatient ctDNA variability over time in EGFR-mutant advanced NSCLC includes changes that may confound interpretation of treatment activity, depending on the magnitude of change used to indicate molecular response. These findings demonstrate that evaluation of on-treatment changes must account for potential background variability, including technical variability because of factors such as cfDNA input level and tumor-related VAF, and that baseline samples should be obtained as close as possible to treatment initiation to minimize the impact of background variability.
Trial registration: ClinicalTrials.gov NCT02296125 NCT02151981.