title: Massively parallel sequencing fails to detect minor resistant subclones in tissue samples prior to tyrosine kinase inhibitor therapy.
abstract: BACKGROUND
  Personalised medicine and targeted therapy have revolutionised cancer treatment. However, most patients develop drug resistance and relapse after showing an initial treatment response. Two theories have been postulated; either secondary resistance mutations develop de novo during therapy by mutagenesis or they are present in minor subclones prior to therapy. In this study, these two theories were evaluated in gastrointestinal stromal tumours (GISTs) where most patients develop secondary resistance mutations in the KIT gene during therapy with tyrosine kinase inhibitors.
  
  
  METHODS
  We used a cohort of 33 formalin-fixed, paraffin embedded (FFPE) primary GISTs and their corresponding recurrent tumours with known mutational status. The primary tumours were analysed for the secondary mutations of the recurrences, which had been identified previously. The primary tumours were resected prior to tyrosine kinase inhibitor therapy. Three ultrasensitive, massively parallel sequencing approaches on the GS Junior (Roche, Mannheim, Germany) and the MiSeq(TM) (Illumina, San Diego, CA, USA) were applied. Additionally, nine fresh-frozen samples resected prior to therapy were analysed for the most common secondary resistance mutations.
  
  
  RESULTS
  With a sensitivity level of down to 0.02%, no pre-existing resistant subclones with secondary KIT mutations were detected in primary GISTs. The sensitivity level varied for individual secondary mutations and was limited by sequencing artefacts on both systems. Artificial T > C substitutions at the position of the exon 13 p.V654A mutation, in particular, led to a lower sensitivity, independent from the source of the material. Fresh-frozen samples showed the same range of artificially mutated allele frequencies as the FFPE material.
  
  
  CONCLUSIONS
  Although we achieved a sufficiently high level of sensitivity, neither in the primary FFPE nor in the fresh-frozen GISTs we were able to detect pre-existing resistant subclones of the corresponding known secondary resistance mutations of the recurrent tumours. This supports the theory that secondary KIT resistance mutations develop under treatment by "de novo" mutagenesis. Alternatively, the detection limit of two mutated clones in 10,000 wild-type clones might not have been high enough or heterogeneous tissue samples, per se, might not be suitable for the detection of very small subpopulations of mutated cells.
authors.affiliation: Institute of Pathology, University Hospital Cologne, Kerpener Str. 62, 50937, Cologne, Germany. carina.heydt@uk-koeln.de.
authors.forename: Carina
authors.initials: C
authors.lastname: Heydt
authors.affiliation: Institute of Pathology, University Hospital Erlangen, Krankenhausstraße 8-10, 91054, Erlangen, Germany. niklas.kumm@web.de.
authors.forename: Niklas
authors.initials: N
authors.lastname: Kumm
authors.affiliation: Institute of Pathology, University Hospital Cologne, Kerpener Str. 62, 50937, Cologne, Germany. jana.fassunke@uk-koeln.de.
authors.forename: Jana
authors.initials: J
authors.lastname: Fassunke
authors.affiliation: Institute of Pathology, University Hospital Cologne, Kerpener Str. 62, 50937, Cologne, Germany. helen.kuenstlinger@uk-koeln.de.
authors.forename: Helen
authors.initials: H
authors.lastname: Künstlinger
authors.affiliation: Institute of Pathology, University Hospital Cologne, Kerpener Str. 62, 50937, Cologne, Germany. michaela.ihle@uk-koeln.de.
authors.forename: Michaela Angelika
authors.initials: MA
authors.lastname: Ihle
authors.affiliation: Institute of Pathology, University Hospital Cologne, Kerpener Str. 62, 50937, Cologne, Germany. andreas.scheel@uk-koeln.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Scheel
authors.affiliation: Institute of Pathology, University Hospital Göttingen, Robert-Koch-Strasse 40, 37075, Göttingen, Germany. hans-ulrich.schildhaus@med.uni-goettingen.de.
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Schildhaus
authors.affiliation: Institute of Pathology, University Hospital Erlangen, Krankenhausstraße 8-10, 91054, Erlangen, Germany. florian.haller@uk-erlangen.de.
authors.forename: Florian
authors.initials: F
authors.lastname: Haller
authors.affiliation: Institute of Pathology, University Hospital Cologne, Kerpener Str. 62, 50937, Cologne, Germany. reinhard.buettner@uk-koeln.de.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Büttner
authors.affiliation: Institute of Pathology, University Hospital Cologne, Kerpener Str. 62, 50937, Cologne, Germany. m.odenthal@uni-koeln.de.
authors.forename: Margarete
authors.initials: M
authors.lastname: Odenthal
authors.affiliation: Gerhard-Domagk-Institute of Pathology, University Hospital Münster, Albert-Schweitzer-Campus 1, Gebäude D17, 48149, Münster, Germany. eva.wardelmann@ukmuenster.de.
authors.forename: Eva
authors.initials: E
authors.lastname: Wardelmann
authors.affiliation: Institute of Pathology, University Hospital Cologne, Kerpener Str. 62, 50937, Cologne, Germany. sabine.merkelbach-bruse@uk-koeln.de.
authors.forename: Sabine
authors.initials: S
authors.lastname: Merkelbach-Bruse
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015331:Cohort Studies; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics*; D005260:Female; D005770:Gastrointestinal Neoplasms / Q000175:diagnosis / Q000188:drug therapy / Q000235:genetics*; D046152:Gastrointestinal Stromal Tumors / Q000175:diagnosis / Q000188:drug therapy / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D047428:Protein Kinase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use*; D012189:Retrospective Studies
chemical_list: D047428:Protein Kinase Inhibitors