title: Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA.
abstract: OBJECTIVE
  Tumor genotyping using cell-free plasma DNA (cfDNA) has the potential to allow noninvasive assessment of tumor biology, yet many existing assays are cumbersome and vulnerable to false-positive results. We sought to determine whether droplet digital PCR (ddPCR) of cfDNA would allow highly specific and quantitative assessment of tumor genotype.
  
  
  METHODS
  ddPCR assays for EGFR, KRAS, and BRAF mutations were developed using plasma collected from patients with advanced lung cancer or melanoma of a known tumor genotype. Sensitivity and specificity were determined using cancers with nonoverlapping genotypes as positive and negative controls. Serial assessment of response and resistance was studied in patients with EGFR-mutant lung cancer on a prospective trial of erlotinib.
  
  
  RESULTS
  We identified a reference range for EGFR L858R and exon 19 deletions in specimens from KRAS-mutant lung cancer, allowing identification of candidate thresholds with high sensitivity and 100% specificity. Received operative characteristic curve analysis of four assays demonstrated an area under the curve in the range of 0.80 to 0.94. Sensitivity improved in specimens with optimal cfDNA concentrations. Serial plasma genotyping of EGFR-mutant lung cancer on erlotinib demonstrated pretreatment detection of EGFR mutations, complete plasma response in most cases, and increasing levels of EGFR T790M emerging before objective progression.
  
  
  CONCLUSIONS
  Noninvasive genotyping of cfDNA using ddPCR demonstrates assay qualities that could allow effective translation into a clinical diagnostic. Serial quantification of plasma genotype allows noninvasive assessment of response and resistance, including detection of resistance mutations up to 16 weeks before radiographic progression.
authors.affiliation: Department of Medical Oncology, Brigham and Women's Hospital & Harvard Medical School Boston, MA.|Department of Medicine, Brigham and Women's Hospital & Harvard Medical School Boston, MA.
authors.forename: Geoffrey R
authors.initials: GR
authors.lastname: Oxnard
authors.affiliation: Department of Medical Oncology, Brigham and Women's Hospital & Harvard Medical School Boston, MA.|Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute, Brigham and Women's Hospital & Harvard Medical School Boston, MA.
authors.forename: Cloud P
authors.initials: CP
authors.lastname: Paweletz
authors.affiliation: Department of Medical Oncology, Brigham and Women's Hospital & Harvard Medical School Boston, MA.|Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute, Brigham and Women's Hospital & Harvard Medical School Boston, MA.
authors.forename: Yanan
authors.initials: Y
authors.lastname: Kuang
authors.affiliation: Department of Medical Oncology, Brigham and Women's Hospital & Harvard Medical School Boston, MA.
authors.forename: Stacy L
authors.initials: SL
authors.lastname: Mach
authors.affiliation: Department of Medical Oncology, Brigham and Women's Hospital & Harvard Medical School Boston, MA.|Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute, Brigham and Women's Hospital & Harvard Medical School Boston, MA.
authors.forename: Allison
authors.initials: A
authors.lastname: O'Connell
authors.affiliation: Department of Medical Oncology, Brigham and Women's Hospital & Harvard Medical School Boston, MA.|Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute, Brigham and Women's Hospital & Harvard Medical School Boston, MA.
authors.forename: Melissa M
authors.initials: MM
authors.lastname: Messineo
authors.affiliation: Department of Medical Oncology, Brigham and Women's Hospital & Harvard Medical School Boston, MA.|Department of Medicine, Brigham and Women's Hospital & Harvard Medical School Boston, MA.
authors.forename: Jason J
authors.initials: JJ
authors.lastname: Luke
authors.affiliation: Department of Medical Oncology, Brigham and Women's Hospital & Harvard Medical School Boston, MA.
authors.forename: Mohit
authors.initials: M
authors.lastname: Butaney
authors.affiliation: Department of Medical Oncology, Brigham and Women's Hospital & Harvard Medical School Boston, MA.|Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute, Brigham and Women's Hospital & Harvard Medical School Boston, MA.
authors.forename: Paul
authors.initials: P
authors.lastname: Kirschmeier
authors.affiliation: Department of Medical Oncology, Brigham and Women's Hospital & Harvard Medical School Boston, MA.|Department of Medicine, Brigham and Women's Hospital & Harvard Medical School Boston, MA.
authors.forename: David M
authors.initials: DM
authors.lastname: Jackman
authors.affiliation: Department of Medical Oncology, Brigham and Women's Hospital & Harvard Medical School Boston, MA.|Belfer Institute for Applied Cancer Science, Dana-Farber Cancer Institute, Brigham and Women's Hospital & Harvard Medical School Boston, MA.|Department of Medicine, Brigham and Women's Hospital & Harvard Medical School Boston, MA.
authors.forename: Pasi A
authors.initials: PA
authors.lastname: Jänne
mesh_terms: D019540:Area Under Curve; D002474:Cell-Free System; D004247:DNA / Q000097:blood*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D066246:ErbB Receptors / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods*; D005838:Genotype; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood / Q000235:genetics*; D009154:Mutation; D010949:Plasma; D016133:Polymerase Chain Reaction / Q000379:methods*; D012372:ROC Curve
chemical_list: D004247:DNA; C512478:EGFR protein, human; D066246:ErbB Receptors