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

<p>Abstract</p> <p>Background</p> <p>Gastrointestinal stromal tumors (GIST) represent the most common mesenchymal tumors of the gastrointestinal tract. About 85% carry an activating mutation in the <it>KIT </it>or <it>PDGFRA </it>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 <it>KIT </it>as well as of the alternative receptor tyrosine kinase genes <it>FLT3</it>, <it>CSF1-R</it>, <it>PDGFRB</it>, <it>AXL </it>and <it>MET </it>by qPCR. wt-GIST were compared to samples with mutations in <it>KIT </it>exon 9 and 11 and <it>PDGFRA </it>exon 18 in order to evaluate whether overexpression of these alternative RTK might contribute to the pathogenesis of wt-GIST.</p> <p>Results</p> <p>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 <it>POLR2A</it>, <it>PPIA</it>, <it>RPLPO </it>and <it>TFRC </it>were chosen for further analysis of the GIST samples. Overexpression of <it>KIT </it>compared to the corresponding normal tissue was detected in each GIST subgroup except in GIST with <it>PDGFRA </it>exon 18 mutation. Comparing our sample groups, no significant differences in the gene expression levels of <it>FLT3</it>, <it>CSF1R </it>and <it>AXL </it>were determined. An exception was the sample group with <it>KIT </it>exon 9 mutation. A significantly reduced expression of <it>CSF1R</it>, <it>FLT3 </it>and <it>PDGFRB </it>compared to the normal tissue was detected. GIST with mutations in <it>KIT </it>exon 9 and 11 and in <it>PDGFRA </it>exon 18 showed a significant <it>PDGFRB </it>downregulation.</p> <p>Conclusions</p> <p>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.</p

The Market for Organic Canned Tomatoes in Germany and the United States

This market research paper has been prepared under the supervision of Prof. Dr. Wolfgang Veit of TH Köln and Prof. Dr. Carol Scovotti of University of Wisconsin-Whitewater in the course of the inter-university cross-border collaboration student research project “Export Opportunity Surveys (EOS)”. This study explores organic canned tomatoes export opportunities to the German and US markets