title: Quantitative assessment of mutant allele burden in solid tumors by semiconductor-based next-generation sequencing.
abstract: OBJECTIVE
  Identification of tumor-specific somatic mutations has had a significant impact on both disease diagnosis and therapy selection. The ability of next-generation sequencing (NGS) to provide a quantitative assessment of mutant allele burden, in numerous target genes in a single assay, provides a significant advantage over conventional qualitative genotyping platforms.
  
  
  METHODS
  We assessed the quantitative capability of NGS and a primer extension-based matrix-assisted laser desorption ionization-time-of-flight (PE-MALDI) assay and directly correlated NGS mutant allele burden determination to morphologic assessment of tumor percentage in H&E-stained slides.
  
  
  RESULTS
  Our results show a 100% concordance between NGS and PE-MALDI in mutant allele detection and a significant correlation between NGS and PE-MALDI for determining mutant allele burden when mutant allele burden is 10% or more.
  
  
  CONCLUSIONS
  NGS-based mutation screening provides a quantitative assessment comparable to that of PE-MALDI. In addition, NGS also allows for a high degree of multiplexing and uses nanogram quantities of DNA, thereby preserving precious material for future analysis. Furthermore, this study provides evidence that H&E-based morphologic assessment of tumor burden does not correlate to actual tumor mutant allele burden frequency.
authors.affiliation: Dept of Hematopathology, MD Anderson Cancer Center, 1515 Holcombe Blvd, Unit 149, Houston, TX 77030; e-mail: kppatel@mdanderson.org.
authors.forename: Bryce P
authors.initials: BP
authors.lastname: Portier
authors.forename: Rashmi
authors.initials: R
authors.lastname: Kanagal-Shamanna
authors.forename: Rajyalakshmi
authors.initials: R
authors.lastname: Luthra
authors.forename: Rajesh
authors.initials: R
authors.lastname: Singh
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Routbort
authors.forename: Brian
authors.initials: B
authors.lastname: Handal
authors.forename: Neelima
authors.initials: N
authors.lastname: Reddy
authors.forename: Bedia A
authors.initials: BA
authors.lastname: Barkoh
authors.forename: Zhuang
authors.initials: Z
authors.lastname: Zuo
authors.forename: L Jeffrey
authors.initials: LJ
authors.lastname: Medeiros
authors.forename: Kenneth
authors.initials: K
authors.lastname: Aldape
authors.forename: Keyur P
authors.initials: KP
authors.lastname: Patel
mesh_terms: D004252:DNA Mutational Analysis; D052138:Genes, Neoplasm; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012666:Semiconductors; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
keywords: AmpliSeq Cancer Panel; Ion Torrent; Massarray; Mutant allele burden; Mutant allele frequency; Next-generation sequencing; Sequenom