title: Clinical next-generation sequencing in patients with non-small cell lung cancer.
abstract: BACKGROUND
  A clinical assay was implemented to perform next-generation sequencing (NGS) of genes commonly mutated in multiple cancer types. This report describes the feasibility and diagnostic yield of this assay in 381 consecutive patients with non-small cell lung cancer (NSCLC).
  
  
  METHODS
  Clinical targeted sequencing of 23 genes was performed with DNA from formalin-fixed, paraffin-embedded (FFPE) tumor tissue. The assay used Agilent SureSelect hybrid capture followed by Illumina HiSeq 2000, MiSeq, or HiSeq 2500 sequencing in a College of American Pathologists-accredited, Clinical Laboratory Improvement Amendments-certified laboratory. Single-nucleotide variants and insertion/deletion events were reported. This assay was performed before methods were developed to detect rearrangements by NGS.
  
  
  RESULTS
  Two hundred nine of all requisitioned samples (55%) were successfully sequenced. The most common reason for not performing the sequencing was an insufficient quantity of tissue available in the blocks (29%). Excisional, endoscopic, and core biopsy specimens were sufficient for testing in 95%, 66%, and 40% of the cases, respectively. The median turnaround time (TAT) in the pathology laboratory was 21 days, and there was a trend of an improved TAT with more rapid sequencing platforms. Sequencing yielded a mean coverage of 1318×. Potentially actionable mutations (ie, predictive or prognostic) were identified in 46% of 209 samples and were most commonly found in KRAS (28%), epidermal growth factor receptor (14%), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (4%), phosphatase and tensin homolog (1%), and BRAF (1%). Five percent of the samples had multiple actionable mutations. A targeted therapy was instituted on the basis of NGS in 11% of the sequenced patients or in 6% of all patients.
  
  
  CONCLUSIONS
  NGS-based diagnostics are feasible in NSCLC and provide clinically relevant information from readily available FFPE tissue. The sample type is associated with the probability of successful testing.
authors.affiliation: Division of Laboratory and Genomic Medicine, Department of Pathology and Immunology, Washington University, St. Louis, Missouri.
authors.forename: Ian S
authors.initials: IS
authors.lastname: Hagemann
authors.forename: Siddhartha
authors.initials: S
authors.lastname: Devarakonda
authors.forename: Christina M
authors.initials: CM
authors.lastname: Lockwood
authors.forename: David H
authors.initials: DH
authors.lastname: Spencer
authors.forename: Kalin
authors.initials: K
authors.lastname: Guebert
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Bredemeyer
authors.forename: Hussam
authors.initials: H
authors.lastname: Al-Kateb
authors.forename: TuDung T
authors.initials: TT
authors.lastname: Nguyen
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Duncavage
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Cottrell
authors.forename: Shashikant
authors.initials: S
authors.lastname: Kulkarni
authors.forename: Rakesh
authors.initials: R
authors.lastname: Nagarajan
authors.forename: Karen
authors.initials: K
authors.lastname: Seibert
authors.forename: Maria
authors.initials: M
authors.lastname: Baggstrom
authors.forename: Saiama N
authors.initials: SN
authors.lastname: Waqar
authors.forename: John D
authors.initials: JD
authors.lastname: Pfeifer
authors.forename: Daniel
authors.initials: D
authors.lastname: Morgensztern
authors.forename: Ramaswamy
authors.initials: R
authors.lastname: Govindan
mesh_terms: D000328:Adult; D000368:Aged; D002289:Carcinoma, Non-Small-Cell Lung / Q000175:diagnosis* / Q000235:genetics*; D004273:DNA, Neoplasm / Q000032:analysis*; D005240:Feasibility Studies; D005260:Female; D005404:Fixatives; D005557:Formaldehyde; D017353:Gene Deletion; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D016254:Mutagenesis, Insertional; D016612:Paraffin Embedding; D020641:Polymorphism, Single Nucleotide; D011518:Proto-Oncogene Proteins / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D017422:Sequence Analysis, DNA / Q000379:methods*; D018631:ras Proteins / Q000235:genetics
chemical_list: D004273:DNA, Neoplasm; D005404:Fixatives; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
keywords: biomarkers; high-throughput nucleotide sequencing; molecular targeted therapy; neoplasms; non-small cell lung cancer; personalized medicine