Detection of ubiquitous and heterogeneous mutations in cell-free DNA from patients with early-stage non-small-cell lung cancer

Abstract

Background: The aim of this pilot study was to assess whether both ubiquitous and heterogeneous somatic mutationscould be detected in cell-free DNA (cfDNA) from patients with early-stage non-small-cell lung cancer (NSCLC).Patients and methods: Three stage I and one stage II primary NSCLC tumors were subjected to multiregion wholeexomesequencing (WES) and validated with AmpliSeq. A subset of ubiquitous and heterogeneous single-nucleotide variants(SNVs) were chosen. Multiplexed PCR using custom-designed primers, coupled with next-generation sequencing(mPCR-NGS), was used to detect these SNVs in both tumor DNA and cfDNA isolated from plasma obtained before surgicalresection of the tumors. The limit of detection for each assay was determined using cfDNA from 48 presumed-normalhealthy volunteers.Results: Tumor DNA and plasma-derived cfDNA was successfully amplified and sequenced for 37/50 (74%) SNVsusing the mPCR-NGS method. Twenty-five (68%) were ubiquitous and 12 (32%) were heterogeneous SNVs. Variant detectionby mPCR-NGS and WES-AmpliSeq in tumor tissue was well correlated (R2 = 0.8722, P < 0.0001). Sixteen (43%)out of 37 SNVs were detected in cfDNA. Twelve of these were ubiquitous SNVs with a variant allele frequency (VAF) rangeof 0.15-23.25%, and four of these were heterogeneous SNVs with a VAF range of 0.28-1.71%. There was a statisticallysignificant linear relationship between the VAFs for tumor and cfDNA (R2 = 0.5144; P = 0.0018). For all four patients, atleast two variants were detected in plasma. The estimated number of copies of variant DNA present in each sampleranged from 5 to 524. The average number of variant copies required for detection (VCRD) was 3.16 (range: 0.2-7.6copies).Conclusions: The mPCR-NGS method revealed intratumor heterogeneity in early-stage NSCLC tumors, and was ableto detect both ubiquitous and heterogeneous SNVs in cfDNA. Further validation of mPCR-NGS in cfDNA is required todefine its potential use in clinical practice.