title: Bias-Corrected Targeted Next-Generation Sequencing for Rapid, Multiplexed Detection of Actionable Alterations in Cell-Free DNA from Advanced Lung Cancer Patients.
abstract: OBJECTIVE
  Tumor genotyping is a powerful tool for guiding non-small cell lung cancer (NSCLC) care; however, comprehensive tumor genotyping can be logistically cumbersome. To facilitate genotyping, we developed a next-generation sequencing (NGS) assay using a desktop sequencer to detect actionable mutations and rearrangements in cell-free plasma DNA (cfDNA).
  
  
  METHODS
  An NGS panel was developed targeting 11 driver oncogenes found in NSCLC. Targeted NGS was performed using a novel methodology that maximizes on-target reads, and minimizes artifact, and was validated on DNA dilutions derived from cell lines. Plasma NGS was then blindly performed on 48 patients with advanced, progressive NSCLC and a known tumor genotype, and explored in two patients with incomplete tumor genotyping.
  
  
  RESULTS
  NGS could identify mutations present in DNA dilutions at ≥ 0.4% allelic frequency with 100% sensitivity/specificity. Plasma NGS detected a broad range of driver and resistance mutations, including ALK, ROS1, and RET rearrangements, HER2 insertions, and MET amplification, with 100% specificity. Sensitivity was 77% across 62 known driver and resistance mutations from the 48 cases; in 29 cases with common EGFR and KRAS mutations, sensitivity was similar to droplet digital PCR. In two cases with incomplete tumor genotyping, plasma NGS rapidly identified a novel EGFR exon 19 deletion and a missed case of MET amplification.
  
  
  CONCLUSIONS
  Blinded to tumor genotype, this plasma NGS approach detected a broad range of targetable genomic alterations in NSCLC with no false positives including complex mutations like rearrangements and unexpected resistance mutations such as EGFR C797S. Through use of widely available vacutainers and a desktop sequencing platform, this assay has the potential to be implemented broadly for patient care and translational research.
authors.affiliation: Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, Massachusetts.
authors.forename: Cloud P
authors.initials: CP
authors.lastname: Paweletz
authors.affiliation: Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.
authors.forename: Adrian G
authors.initials: AG
authors.lastname: Sacher
authors.affiliation: Resolution Bioscience, Bellevue, Washington.
authors.forename: Chris K
authors.initials: CK
authors.lastname: Raymond
authors.affiliation: Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.
authors.forename: Ryan S
authors.initials: RS
authors.lastname: Alden
authors.affiliation: Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, Massachusetts.
authors.forename: Allison
authors.initials: A
authors.lastname: O'Connell
authors.affiliation: Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.
authors.forename: Stacy L
authors.initials: SL
authors.lastname: Mach
authors.affiliation: Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, Massachusetts.
authors.forename: Yanan
authors.initials: Y
authors.lastname: Kuang
authors.affiliation: Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.
authors.forename: Leena
authors.initials: L
authors.lastname: Gandhi
authors.affiliation: Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, Massachusetts.
authors.forename: Paul
authors.initials: P
authors.lastname: Kirschmeier
authors.affiliation: Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, Massachusetts.
authors.forename: Jessie M
authors.initials: JM
authors.lastname: English
authors.affiliation: Resolution Bioscience, Bellevue, Washington.
authors.forename: Lee P
authors.initials: LP
authors.lastname: Lim
authors.affiliation: Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, Massachusetts. Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.
authors.forename: Pasi A
authors.initials: PA
authors.lastname: Jänne
authors.affiliation: Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts. geoffrey_oxnard@dfci.harvard.edu.
authors.forename: Geoffrey R
authors.initials: GR
authors.lastname: Oxnard
mesh_terms: D002289:Carcinoma, Non-Small-Cell Lung / Q000097:blood* / Q000235:genetics / Q000556:secondary; D045744:Cell Line, Tumor; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000097:blood* / Q000235:genetics; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood* / Q000235:genetics / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging
chemical_list: D004273:DNA, Neoplasm