title: qPCR in gastrointestinal stromal tumors: Evaluation of reference genes and expression analysis of KIT and the alternative receptor tyrosine kinases FLT3, CSF1-R, PDGFRB, MET and AXL.
abstract: BACKGROUND
  Gastrointestinal stromal tumors (GIST) represent the most common mesenchymal tumors of the gastrointestinal tract. About 85% carry an activating mutation in the KIT or PDGFRA gene. Approximately 10% of GIST are so-called wild type GIST (wt-GIST) without mutations in the hot spots. In the present study we evaluated appropriate reference genes for the expression analysis of formalin-fixed, paraffin-embedded and fresh frozen samples from gastrointestinal stromal tumors. We evaluated the gene expression of KIT as well as of the alternative receptor tyrosine kinase genes FLT3, CSF1-R, PDGFRB, AXL and MET by qPCR. wt-GIST were compared to samples with mutations in KIT exon 9 and 11 and PDGFRA exon 18 in order to evaluate whether overexpression of these alternative RTK might contribute to the pathogenesis of wt-GIST.
  
  
  RESULTS
  Gene expression variability of the pooled cDNA samples is much lower than the single reverse transcription cDNA synthesis. By combining the lowest variability values of fixed and fresh tissue, the genes POLR2A, PPIA, RPLPO and TFRC were chosen for further analysis of the GIST samples. Overexpression of KIT compared to the corresponding normal tissue was detected in each GIST subgroup except in GIST with PDGFRA exon 18 mutation. Comparing our sample groups, no significant differences in the gene expression levels of FLT3, CSF1R and AXL were determined. An exception was the sample group with KIT exon 9 mutation. A significantly reduced expression of CSF1R, FLT3 and PDGFRB compared to the normal tissue was detected. GIST with mutations in KIT exon 9 and 11 and in PDGFRA exon 18 showed a significant PDGFRB downregulation.
  
  
  CONCLUSIONS
  As the variability of expression levels for the reference genes is very high comparing fresh frozen and formalin-fixed tissue there is a strong need for validation in each tissue type. None of the alternative receptor tyrosine kinases analyzed is associated with the pathogenesis of wild-type or mutated GIST. It remains to be clarified whether an autocrine or paracrine mechanism by overexpression of receptor tyrosine kinase ligands is responsible for the tumorigenesis of wt-GIST.
authors.affiliation: Department of Pathology, University of Bonn Medical Center, Bonn, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Fassunke
authors.forename: Marie-Christine
authors.initials: MC
authors.lastname: Blum
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Schildhaus
authors.forename: Marc
authors.initials: M
authors.lastname: Zapatka
authors.forename: Benedikt
authors.initials: B
authors.lastname: Brors
authors.forename: Helen
authors.initials: H
authors.lastname: Künstlinger
authors.forename: Reinhard
authors.initials: R
authors.lastname: Büttner
authors.forename: Eva
authors.initials: E
authors.lastname: Wardelmann
authors.forename: Sabine
authors.initials: S
authors.lastname: Merkelbach-Bruse
mesh_terms: D018076:DNA, Complementary / Q000235:genetics; D046152:Gastrointestinal Stromal Tumors / Q000235:genetics* / Q000473:pathology; D020869:Gene Expression Profiling / Q000592:standards; D015972:Gene Expression Regulation, Neoplastic; D005796:Genes; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000592:standards; D011518:Proto-Oncogene Proteins / Q000235:genetics; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics*; D019859:Proto-Oncogene Proteins c-met / Q000235:genetics; D012313:RNA / Q000302:isolation & purification; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000235:genetics; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000235:genetics; D012015:Reference Standards; D051941:fms-Like Tyrosine Kinase 3 / Q000235:genetics; D000094963:Axl Receptor Tyrosine Kinase
chemical_list: D018076:DNA, Complementary; D011518:Proto-Oncogene Proteins; D012313:RNA; C497970:FLT3 protein, human; D019009:Proto-Oncogene Proteins c-kit; D019859:Proto-Oncogene Proteins c-met; D020794:Receptor Protein-Tyrosine Kinases; D016186:Receptor, Macrophage Colony-Stimulating Factor; D020797:Receptor, Platelet-Derived Growth Factor beta; D051941:fms-Like Tyrosine Kinase 3; D000094963:Axl Receptor Tyrosine Kinase; C000722289:AXL protein, human