title: Prospective Validation of Rapid Plasma Genotyping for the Detection of EGFR and KRAS Mutations in Advanced Lung Cancer.
abstract: OBJECTIVE
  Plasma genotyping of cell-free DNA has the potential to allow for rapid noninvasive genotyping while avoiding the inherent shortcomings of tissue genotyping and repeat biopsies.
  
  
  OBJECTIVE
  To prospectively validate plasma droplet digital PCR (ddPCR) for the rapid detection of common epidermal growth factor receptor (EGFR) and KRAS mutations, as well as the EGFR T790M acquired resistance mutation.
  
  
  METHODS
  Patients with advanced nonsquamous non-small-cell lung cancer (NSCLC) who either (1) had a new diagnosis and were planned for initial therapy or (2) had developed acquired resistance to an EGFR kinase inhibitor and were planned for rebiopsy underwent initial blood sampling and immediate plasma ddPCR for EGFR exon 19 del, L858R, T790M, and/or KRAS G12X between July 3, 2014, and June 30, 2015, at a National Cancer Institute-designated comprehensive cancer center. All patients underwent biopsy for tissue genotyping, which was used as the reference standard for comparison; rebiopsy was required for patients with acquired resistance to EGFR kinase inhibitors. Test turnaround time (TAT) was measured in business days from blood sampling until test reporting.
  
  
  METHODS
  Plasma ddPCR assay sensitivity, specificity, and TAT.
  
  
  RESULTS
  Of 180 patients with advanced NSCLC (62% female; median [range] age, 62 [37-93] years), 120 cases were newly diagnosed; 60 had acquired resistance. Tumor genotype included 80 EGFR exon 19/L858R mutants, 35 EGFR T790M, and 25 KRAS G12X mutants. Median (range) TAT for plasma ddPCR was 3 (1-7) days. Tissue genotyping median (range) TAT was 12 (1-54) days for patients with newly diagnosed NSCLC and 27 (1-146) days for patients with acquired resistance. Plasma ddPCR exhibited a positive predictive value of 100% (95% CI, 91%-100%) for EGFR 19 del, 100% (95% CI, 85%-100%) for L858R, and 100% (95% CI, 79%-100%) for KRAS, but lower for T790M at 79% (95% CI, 62%-91%). The sensitivity of plasma ddPCR was 82% (95% CI, 69%-91%) for EGFR 19 del, 74% (95% CI, 55%-88%) for L858R, and 77% (95% CI, 60%-90%) for T790M, but lower for KRAS at 64% (95% CI, 43%-82%). Sensitivity for EGFR or KRAS was higher in patients with multiple metastatic sites and those with hepatic or bone metastases, specifically.
  
  
  CONCLUSIONS
  Plasma ddPCR detected EGFR and KRAS mutations rapidly with the high specificity needed to select therapy and avoid repeat biopsies. This assay may also detect EGFR T790M missed by tissue genotyping due to tumor heterogeneity in resistant disease.
authors.affiliation: Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts2Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts.
authors.forename: Adrian G
authors.initials: AG
authors.lastname: Sacher
authors.affiliation: Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, Massachusetts.
authors.forename: Cloud
authors.initials: C
authors.lastname: Paweletz
authors.affiliation: Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Boston, Massachusetts5Harvard T.H. Chan School of Public Health, Boston, Massachusetts.
authors.forename: Suzanne E
authors.initials: SE
authors.lastname: Dahlberg
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: Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, Massachusetts.
authors.forename: Nora
authors.initials: N
authors.lastname: Feeney
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: Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts2Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts3Belfer Center for Applied Cancer Science, Dana-Farber Cancer Institute, Boston, Massachus.
authors.forename: Pasi A
authors.initials: PA
authors.lastname: Jänne
authors.affiliation: Lowe Center for Thoracic Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts2Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts.
authors.forename: Geoffrey R
authors.initials: GR
authors.lastname: Oxnard
mesh_terms: D000230:Adenocarcinoma / Q000175:diagnosis / Q000188:drug therapy / Q000235:genetics*; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000970:Antineoplastic Agents / Q000627:therapeutic use; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis / Q000188:drug therapy / Q000235:genetics*; D000066491:Clinical Decision-Making; D004273:DNA, Neoplasm / Q000032:analysis / Q000097:blood*; D019008:Drug Resistance, Neoplasm; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics*; D000069347:Erlotinib Hydrochloride / Q000627:therapeutic use; D005260:Female; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis / Q000188:drug therapy / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D057285:Precision Medicine; D011446:Prospective Studies; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D000970:Antineoplastic Agents; D004273:DNA, Neoplasm; C117307:KRAS protein, human; D047428:Protein Kinase Inhibitors; D000069347:Erlotinib Hydrochloride; C512478:EGFR protein, human; D066246:ErbB Receptors; D016283:Proto-Oncogene Proteins p21(ras)