Clinical Next-Generation Sequencing in Patients With Non-Small Cell Lung Cancer

Cancer. 2015 Feb 15;121(4):631-9. doi: 10.1002/cncr.29089. Epub 2014 Oct 24.

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.

Keywords: biomarkers; high-throughput nucleotide sequencing; molecular targeted therapy; neoplasms; non-small cell lung cancer; personalized medicine.

MeSH terms

  • Adult
  • Aged
  • Carcinoma, Non-Small-Cell Lung / diagnosis*
  • Carcinoma, Non-Small-Cell Lung / genetics*
  • DNA, Neoplasm / analysis*
  • Feasibility Studies
  • Female
  • Fixatives
  • Formaldehyde
  • Gene Deletion*
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Lung Neoplasms / diagnosis*
  • Lung Neoplasms / genetics*
  • Male
  • Middle Aged
  • Mutagenesis, Insertional*
  • Paraffin Embedding
  • Polymorphism, Single Nucleotide*
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins B-raf / genetics
  • Proto-Oncogene Proteins p21(ras)
  • Sequence Analysis, DNA / methods*
  • ras Proteins / genetics

Substances

  • DNA, Neoplasm
  • Fixatives
  • KRAS protein, human
  • Proto-Oncogene Proteins
  • Formaldehyde
  • BRAF protein, human
  • Proto-Oncogene Proteins B-raf
  • Proto-Oncogene Proteins p21(ras)
  • ras Proteins