%0 Journal Article %1 Fassunke2010 %A Fassunke, Jana %A Blum, Marie-Christine %A Schildhaus, Hans-Ulrich %A Zapatka, Marc %A Brors, Benedikt %A Künstlinger, Helen %A Büttner, Reinhard %A Wardelmann, Eva %A Merkelbach-Bruse, Sabine %D 2010 %J BMC Mol Biol %K /&/ 3, Chain Colony-Stimulating Complementary, DNA, Expression Factor Factor, Gastrointestinal Gene Genes; Growth Humans; Kinase Kinases, Macrophage Mutation; Neoplastic; Platelet-Derived Polymerase Profiling, Protein-Tyrosine Proteins Proteins, Proto-Oncogene RNA, Reaction, Receptor Receptor, Reference Regulation, Standards; Stromal Tumors, Tyrosine beta, c-kit, c-met, fms-Like genetics genetics/pathology; genetics; isolation purification; standards; %P 100 %R 10.1186/1471-2199-11-100 %T 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. %U http://dx.doi.org/10.1186/1471-2199-11-100 %V 11 %X 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.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.

@article{Fassunke2010, __markedentry = {[bbrors:6]}, abstract = {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.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.}, added-at = {2015-04-09T12:36:21.000+0200}, author = {Fassunke, Jana and Blum, Marie-Christine and Schildhaus, Hans-Ulrich and Zapatka, Marc and Brors, Benedikt and K{\"{u}}nstlinger, Helen and B{\"{u}}ttner, Reinhard and Wardelmann, Eva and Merkelbach-Bruse, Sabine}, biburl = {https://www.bibsonomy.org/bibtex/2495f8ab2c3fd02354dd179ab270c600f/bbrors}, doi = {10.1186/1471-2199-11-100}, institution = {Department of Pathology, University of Bonn Medical Center, Bonn, Germany.}, interhash = {4172b23096a3d35f9f91d9287ac892f7}, intrahash = {495f8ab2c3fd02354dd179ab270c600f}, journal = {BMC Mol Biol}, keywords = {/&/ 3, Chain Colony-Stimulating Complementary, DNA, Expression Factor Factor, Gastrointestinal Gene Genes; Growth Humans; Kinase Kinases, Macrophage Mutation; Neoplastic; Platelet-Derived Polymerase Profiling, Protein-Tyrosine Proteins Proteins, Proto-Oncogene RNA, Reaction, Receptor Receptor, Reference Regulation, Standards; Stromal Tumors, Tyrosine beta, c-kit, c-met, fms-Like genetics genetics/pathology; genetics; isolation purification; standards;}, language = {eng}, medline-pst = {epublish}, owner = {bbrors}, pages = 100, pii = {1471-2199-11-100}, pmid = {21171987}, timestamp = {2015-04-09T12:36:21.000+0200}, 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.}, url = {http://dx.doi.org/10.1186/1471-2199-11-100}, volume = 11, year = 2010 }