title: Detection of ubiquitous and heterogeneous mutations in cell-free DNA from patients with early-stage non-small-cell lung cancer.
abstract: 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, 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) 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.
authors.affiliation: Translational Cancer Therapeutics Laboratory, Cancer Research UK Lung Cancer Centre of Excellence, University College London Cancer Institute, London.
authors.forename: M
authors.initials: M
authors.lastname: Jamal-Hanjani
authors.affiliation: Translational Cancer Therapeutics Laboratory, Cancer Research UK Lung Cancer Centre of Excellence, University College London Cancer Institute, London Translational Cancer Therapeutics Laboratory, The Francis Crick Institute, London.
authors.forename: G A
authors.initials: GA
authors.lastname: Wilson
authors.affiliation: Department of Bioinformatics and Biostatistics, The Francis Crick Institute, London, UK.
authors.forename: S
authors.initials: S
authors.lastname: Horswell
authors.affiliation: Department of Bioinformatics and Biostatistics, The Francis Crick Institute, London, UK.
authors.forename: R
authors.initials: R
authors.lastname: Mitter
authors.affiliation: Department of Bioinformatics and Biostatistics, The Francis Crick Institute, London, UK.
authors.forename: O
authors.initials: O
authors.lastname: Sakarya
authors.affiliation: Natera Inc., San Carlos, USA.
authors.forename: T
authors.initials: T
authors.lastname: Constantin
authors.affiliation: Natera Inc., San Carlos, USA.
authors.forename: R
authors.initials: R
authors.lastname: Salari
authors.affiliation: Natera Inc., San Carlos, USA.
authors.forename: E
authors.initials: E
authors.lastname: Kirkizlar
authors.affiliation: Natera Inc., San Carlos, USA.
authors.forename: S
authors.initials: S
authors.lastname: Sigurjonsson
authors.affiliation: Natera Inc., San Carlos, USA.
authors.forename: R
authors.initials: R
authors.lastname: Pelham
authors.affiliation: Natera Inc., San Carlos, USA.
authors.forename: S
authors.initials: S
authors.lastname: Kareht
authors.affiliation: Natera Inc., San Carlos, USA.
authors.forename: B
authors.initials: B
authors.lastname: Zimmermann
authors.affiliation: Translational Cancer Therapeutics Laboratory, Cancer Research UK Lung Cancer Centre of Excellence, University College London Cancer Institute, London Translational Cancer Therapeutics Laboratory, The Francis Crick Institute, London charles.swanton@crick.ac.uk.
authors.forename: C
authors.initials: C
authors.lastname: Swanton
mesh_terms: D000368:Aged; D002289:Carcinoma, Non-Small-Cell Lung / Q000097:blood / Q000235:genetics* / Q000473:pathology; D000073888:Cell-Free Nucleic Acids / Q000097:blood / Q000235:genetics*; D004273:DNA, Neoplasm / Q000097:blood / Q000235:genetics*; D005260:Female; D018740:Genetic Heterogeneity; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009367:Neoplasm Staging; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D000073359:Exome Sequencing
chemical_list: D000073888:Cell-Free Nucleic Acids; D004273:DNA, Neoplasm
keywords: circulating cell-free DNA; clonal; intratumor heterogeneity; non-small-cell lung cancer; subclonal