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The aim of this pilot study was to assess whether both ubiquitous and heterogeneous somatic mutations could be detected in cell-free DNA (cfDNA) from patients with early-stage non-small-cell lung cancer (NSCLC).Three stage I and one stage II primary NSCLC tumors were subjected to multiregion whole-exome sequencing (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 surgical resection of the tumors. The limit of detection for each assay was determined using cfDNA from 48 presumed-normal healthy volunteers.Tumor DNA and plasma-derived cfDNA was successfully amplified and sequenced for 37/50 (74%) SNVs using the mPCR-NGS method. Twenty-five (68%) were ubiquitous and 12 (32%) were heterogeneous SNVs. Variant detection by mPCR-NGS and WES-AmpliSeq in tumor tissue was well correlated (R(2) = 0.8722, P0.0001). Sixteen (43%) out of 37 SNVs were detected in cfDNA. Twelve of these were ubiquitous SNVs with a variant allele frequency (VAF) range of 0.15-23.25%, and four of these were heterogeneous SNVs with a VAF range of 0.28-1.71%. There was a statistically significant linear relationship between the VAFs for tumor and cfDNA (R(2) = 0.5144; P = 0.0018). For all four patients, at least two variants were detected in plasma. The estimated number of copies of variant DNA present in each sample ranged from 5 to 524. The average number of variant copies required for detection (VCRD) was 3.16 (range: 0.2-7.6 copies).The mPCR-NGS method revealed intratumor heterogeneity in early-stage NSCLC tumors, and was able to detect both ubiquitous and heterogeneous SNVs in cfDNA. Further validation of mPCR-NGS in cfDNA is required to define its potential use in clinical practice.

Aim: Few studies have investigated the clonal architecture of non-small cell lung cancer (NSCLC) and its evolution over time. With increasing evidence for the potential impact of intratumour heterogeneity (ITH) on clinical outcome, and its recognition as a driver of cancer progression, there is an unmet need to elicit the implications of ITH for biomarker validation, therapeutic response, and therefore survival. Methods: In a cohort of 11 primary NSCLC tumours, multi-region whole-exome sequencing at a mean depth of 214x was performed on a total of 37 tumour regions. Single nucleotide variants, indels and copy number data were identified using VarScan2 (v2.3.6). Non-silent mutations were validated using ultra-deep amplicon sequencing (mean depth 1189x). The subclonal composition of each tumor was used to construct phylogenetic trees. Candidate driver genes were selected by referencing large-scale lung cancer or pan-cancer sequencing studies, and the Catalogue of Somatic Mutations in Cancer (COSMIC) gene census. Results: ITH was evident in all tumours, with a median of 33% heterogeneous mutations (range 5–61%). Phylogenetic tree analysis revealed branched tumour evolution, with mutations in potential driver genes occurring both early (trunk) and late (branch). Thirty-five percent (range 8-67%) of mutations in potential driver genes were heterogeneous, and included TP53, EGFR, KRAS, BRAF, ALK and ROS1. Overall, heterogeneous non-silent somatic mutations and copy number aberrations occurred frequently. Conclusions: This study demonstrates spatial and temporal heterogeneity of both driver mutations and copy number events, suggesting that NSCLCs may follow multiple distinct evolutionary pathways simultaneously. Knowledge of these pathways may help identify novel therapeutic targets to improve clinical outcomes, and large-scale longitudinal genomic studies, such as TRACERx (TRAcking NSCLC Evolution through therapy (Rx), NCT01888601), may become a central component to the delivery of precision cancer medicine. Disclosure: All authors have declared no conflicts of interest.