title: The Interlaboratory RObustness of Next-generation sequencing (IRON) study: a deep sequencing investigation of TET2, CBL and KRAS mutations by an international consortium involving 10 laboratories.
abstract: Massively parallel pyrosequencing allows sensitive deep sequencing to detect molecular aberrations. Thus far, data are limited on the technical performance in a clinical diagnostic setting. Here, we investigated as an international consortium the robustness, precision and reproducibility of amplicon next-generation deep sequencing across 10 laboratories in eight countries. In a cohort of 18 chronic myelomonocytic leukemia patients, mutational analyses were performed on TET2, a frequently mutated gene in myeloproliferative neoplasms. Additionally, hotspot regions of CBL and KRAS were investigated. The study was executed using GS FLX sequencing instruments and the small volume 454 Life Sciences Titanium emulsion PCR setup. We report a high concordance in mutation detection across all laboratories, including a robust detection of novel variants, which were undetected by standard Sanger sequencing. The sensitivity to detect low-level variants present with as low as 1-2% frequency, compared with the 20% threshold for Sanger-based sequencing is increased. Together with the output of high-quality long reads and fast run time, we demonstrate the utility of deep sequencing in clinical applications. In conclusion, this multicenter analysis demonstrated that amplicon-based deep sequencing is technically feasible, achieves high concordance across multiple laboratories and allows a broad and in-depth molecular characterization of cancer specimens with high diagnostic sensitivity.
authors.affiliation: Department of Molecular Genetics, MLL Munich Leukemia Laboratory, Munich, Germany. alexander.kohlmann@mll.com
authors.forename: A
authors.initials: A
authors.lastname: Kohlmann
authors.forename: H-U
authors.initials: HU
authors.lastname: Klein
authors.forename: S
authors.initials: S
authors.lastname: Weissmann
authors.forename: S
authors.initials: S
authors.lastname: Bresolin
authors.forename: T
authors.initials: T
authors.lastname: Chaplin
authors.forename: H
authors.initials: H
authors.lastname: Cuppens
authors.forename: E
authors.initials: E
authors.lastname: Haschke-Becher
authors.forename: B
authors.initials: B
authors.lastname: Garicochea
authors.forename: V
authors.initials: V
authors.lastname: Grossmann
authors.forename: B
authors.initials: B
authors.lastname: Hanczaruk
authors.forename: K
authors.initials: K
authors.lastname: Hebestreit
authors.forename: C
authors.initials: C
authors.lastname: Gabriel
authors.forename: I
authors.initials: I
authors.lastname: Iacobucci
authors.forename: J H
authors.initials: JH
authors.lastname: Jansen
authors.forename: G
authors.initials: G
authors.lastname: te Kronnie
authors.forename: L
authors.initials: L
authors.lastname: van de Locht
authors.forename: G
authors.initials: G
authors.lastname: Martinelli
authors.forename: K
authors.initials: K
authors.lastname: McGowan
authors.forename: M R
authors.initials: MR
authors.lastname: Schweiger
authors.forename: B
authors.initials: B
authors.lastname: Timmermann
authors.forename: P
authors.initials: P
authors.lastname: Vandenberghe
authors.forename: B D
authors.initials: BD
authors.lastname: Young
authors.forename: M
authors.initials: M
authors.lastname: Dugas
authors.forename: T
authors.initials: T
authors.lastname: Haferlach
mesh_terms: D000368:Aged; D000369:Aged, 80 and over; D015331:Cohort Studies; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics*; D049308:Dioxygenases; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D015477:Leukemia, Myelomonocytic, Chronic / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011379:Prognosis; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D050721:Proto-Oncogene Proteins c-cbl / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins / Q000235:genetics*
chemical_list: D004268:DNA-Binding Proteins; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D049308:Dioxygenases; C541081:TET2 protein, human; D050721:Proto-Oncogene Proteins c-cbl; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins; C067419:CBL protein, human