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

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.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.Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract and are characterized by the expression of the KIT receptor (stem cell factor receptor, CD117) and to a lesser extent of PDGFRA (platelet derived growth factor receptor alpha), representing two closely related receptor tyrosine kinases (RTK) [1,2]. The majority of GIST shows oncogenic mutations either in KIT or PDGFRA [3,4]. Mainly, mutations in exon 9 or 11 of the KIT gene or in exon 18 of PDGFRA lead to ligand independent, constitutive activation of the kinase function [5]. About 60% of all GIST carry an exon 11 mutation of KIT which encodes the juxtamembrane domain of th