title: Benchmarking of mutation diagnostics in clinical lung cancer specimens.
abstract: Treatment of EGFR-mutant non-small cell lung cancer patients with the tyrosine kinase inhibitors erlotinib or gefitinib results in high response rates and prolonged progression-free survival. Despite the development of sensitive mutation detection approaches, a thorough validation of these in a clinical setting has so far been lacking. We performed, in a clinical setting, a systematic validation of dideoxy 'Sanger' sequencing and pyrosequencing against massively parallel sequencing as one of the most sensitive mutation detection technologies available. Mutational annotation of clinical lung tumor samples revealed that of all patients with a confirmed response to EGFR inhibition, only massively parallel sequencing detected all relevant mutations. By contrast, dideoxy sequencing missed four responders and pyrosequencing missed two responders, indicating a dramatic lack of sensitivity of dideoxy sequencing, which is widely applied for this purpose. Furthermore, precise quantification of mutant alleles revealed a low correlation (r(2) = 0.27) of histopathological estimates of tumor content and frequency of mutant alleles, thereby questioning the use of histopathology for stratification of specimens for individual analytical procedures. Our results suggest that enhanced analytical sensitivity is critically required to correctly identify patients responding to EGFR inhibition. More broadly, our results emphasize the need for thorough evaluation of all mutation detection approaches against massively parallel sequencing as a prerequisite for any clinical implementation.
authors.affiliation: Max Planck Institute for Neurological Research with Klaus-Joachim-Zülch Laboratories of the Max Planck Society and the Medical Faculty of the University of Cologne, Cologne, Germany.
authors.forename: Silvia
authors.initials: S
authors.lastname: Querings
authors.forename: Janine
authors.initials: J
authors.lastname: Altmüller
authors.forename: Sascha
authors.initials: S
authors.lastname: Ansén
authors.forename: Thomas
authors.initials: T
authors.lastname: Zander
authors.forename: Danila
authors.initials: D
authors.lastname: Seidel
authors.forename: Franziska
authors.initials: F
authors.lastname: Gabler
authors.forename: Martin
authors.initials: M
authors.lastname: Peifer
authors.forename: Eva
authors.initials: E
authors.lastname: Markert
authors.forename: Kathryn
authors.initials: K
authors.lastname: Stemshorn
authors.forename: Bernd
authors.initials: B
authors.lastname: Timmermann
authors.forename: Beate
authors.initials: B
authors.lastname: Saal
authors.forename: Stefan
authors.initials: S
authors.lastname: Klose
authors.forename: Karen
authors.initials: K
authors.lastname: Ernestus
authors.forename: Matthias
authors.initials: M
authors.lastname: Scheffler
authors.forename: Walburga
authors.initials: W
authors.lastname: Engel-Riedel
authors.forename: Erich
authors.initials: E
authors.lastname: Stoelben
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Brambilla
authors.forename: Jürgen
authors.initials: J
authors.lastname: Wolf
authors.forename: Peter
authors.initials: P
authors.lastname: Nürnberg
authors.forename: Roman K
authors.initials: RK
authors.lastname: Thomas
mesh_terms: D000328:Adult; D000368:Aged; D001483:Base Sequence; D019985:Benchmarking; D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy / Q000235:genetics / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods* / Q000592:standards*; D066246:ErbB Receptors / Q000235:genetics; D005091:Exons / Q000235:genetics; D005260:Female; D015894:Genome, Human / Q000235:genetics; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D016896:Treatment Outcome
chemical_list: D066246:ErbB Receptors