title: Identification of Factors Affecting the Success of Next-Generation Sequencing Testing in Solid Tumors.
abstract: OBJECTIVE
  Clinical laboratories are rapidly implementing next-generation sequencing (NGS) tests for mutation analysis, but there are few guidelines regarding sample quality for successful results.
  
  
  METHODS
  We aimed to establish tissue quality parameters for successful NGS in solid tumors and to improve NGS performance.
  
  
  RESULTS
  Analysis of 614 clinical cases tested in 2013 using a 50-gene hotspot mutation panel identified the major cause for unsuccessful NGS analysis was DNA less than 10 ng (91%, 67/74) associated with extremely small and low cellularity samples. High success rates were associated with resection procedures (333/342, 97%) and biopsied tumor larger than 10 mm(2) (77/77, 100%). NGS can be successfully performed on bone specimens processed with formic acid-based decalcification procedures (8/11, 73%). Tumor type and paraffin block age did not affect success. We demonstrated that NGS can be carried out on samples with less than 10 ng DNA. Analysis of 408 cases tested in 2014 using an optimized workflow showed improved NGS success rates from 88% to 95% (387/408) with pronounced improvement among tiny (<10 mm(2)) samples (from 76% to 94%) as well as cytology samples (from 58% to 87%).
  
  
  CONCLUSIONS
  Identifying preanalytical tissue factors allows us to improve NGS performance and to successfully test tumors obtained from minimally invasive procedures.
authors.affiliation: From the Departments of Hematopathology.
authors.forename: Rashmi S
authors.initials: RS
authors.lastname: Goswami
authors.affiliation: From the Departments of Hematopathology rluthra@mdanderson.org.
authors.forename: Rajyalakshmi
authors.initials: R
authors.lastname: Luthra
authors.affiliation: From the Departments of Hematopathology.
authors.forename: Rajesh R
authors.initials: RR
authors.lastname: Singh
authors.affiliation: From the Departments of Hematopathology.
authors.forename: Keyur P
authors.initials: KP
authors.lastname: Patel
authors.affiliation: From the Departments of Hematopathology.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Routbort
authors.affiliation: Pathology.
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Aldape
authors.affiliation: Bioinformatics and Computational Biology, University of Texas MD Anderson Cancer Center, Houston.
authors.forename: Hui
authors.initials: H
authors.lastname: Yao
authors.affiliation: From the Departments of Hematopathology.
authors.forename: Hyvan D
authors.initials: HD
authors.lastname: Dang
authors.affiliation: From the Departments of Hematopathology.
authors.forename: Bedia A
authors.initials: BA
authors.lastname: Barkoh
authors.affiliation: From the Departments of Hematopathology.
authors.forename: Jawad
authors.initials: J
authors.lastname: Manekia
authors.affiliation: From the Departments of Hematopathology.
authors.forename: L Jeffrey
authors.initials: LJ
authors.lastname: Medeiros
authors.affiliation: Pathology.
authors.forename: Sinchita
authors.initials: S
authors.lastname: Roy-Chowdhuri
authors.affiliation: Pathology.
authors.forename: John
authors.initials: J
authors.lastname: Stewart
authors.affiliation: Pathology.
authors.forename: Russell R
authors.initials: RR
authors.lastname: Broaddus
authors.affiliation: Pathology.
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics*; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000032:analysis* / Q000302:isolation & purification; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics; D016612:Paraffin Embedding; D017422:Sequence Analysis, DNA; D013048:Specimen Handling; D016707:Tissue Fixation; D057188:Workflow
chemical_list: D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm
keywords: Ion Torrent; Next-generation sequencing; Preanalytical tissue factors; Targeted hotspot mutation; Tissue qualification