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

MET gene copy number alterations and expression of MET and hepatocyte growth factor are potential biomarkers in angiosarcomas and undifferentiated pleomorphic sarcomas.

Soft tissue sarcomas are a heterogeneous group of tumors with many different subtypes. In 2014 an estimated 12,020 newly diagnosed cases and 4,740 soft tissue sarcoma related deaths can be expected in the United States. Many soft tissue sarcomas are associated with poor prognosis and therapeutic options are often limited. The evolution of precision medicine has not yet fully reached the clinical treatment of sarcomas since therapeutically tractable genetic changes have not been comprehensively studied so far. We analyzed a total of 484 adult-type malignant mesenchymal tumors by MET fluorescence in situ hybridization and MET and hepatocyte growth factor immunohistochemistry. Eleven different entities were included, among them the most common and clinically relevant subtypes and tumors with specific translocations or complex genetic changes. MET protein expression was observed in 2.6% of the cases, all of which were either undifferentiated pleomorphic sarcomas or angiosarcomas, showing positivity rates of 14% and 17%, respectively. 6% of the tumors showed hepatocyte growth factor overexpression, mainly seen in undifferentiated pleomorphic sarcomas and angiosarcomas, but also in clear cell sarcomas, malignant peripheral nerve sheath tumors, leiomyosarcomas and gastrointestinal stromal tumors. MET and hepatocyte growth factor overexpression were significantly correlated and may suggest an autocrine activation in these tumors. MET FISH amplification and copy number gain were present in 4% of the tumors (15/413). Two samples, both undifferentiated pleomorphic sarcomas, fulfilled the criteria for high level amplification of MET, one undifferentiated pleomorphic sarcoma reached an intermediate level copy number gain, and 12 samples of different subtypes were categorized as low level copy number gains for MET. Our findings indicate that angiosarcomas and undifferentiated pleomorphic sarcomas rather than other frequent adult-type sarcomas should be enrolled in screening programs for clinical trials with MET inhibitors. The screening methods should include both in situ hybridization and immunohistochemistry

Comparison of five automated DNA extraction systems.

<p>Illustration of relative DNA concentrations of samples 1–10 measured by the NanoDrop 2000c spectrophotometer (A) and Qubit 2.0 fluorometer (B). The concentrations of the Maxwell 16 extracts were set to 100% for each sample.</p

Results of the five DNA quantification measurements.

<p>Distribution of DNA concentrations of samples 1–16 measured by each of the five quantification method.</p

Impact of DNA quantification methods on massively parallel sequencing.

<p>(A) Mean amplicon coverage determined by in-house software for each sample, quantification method and amount of DNA used for multiplex PCR amplification. (B) Number of all variants called by in-house software for each sample, quantification method and starting material used.</p

Impact of DNA quantification methods on downstream applications.

<p>(A) Electrophoretic pattern of 13 DNA extracts on a 1% agarose gel. (B) Amplified 275 bp fragment of the <i>EGFR</i> gene. 20 ng of sample DNA determined by each quantification method was used for PCR amplification. + indicates a positive control, - a negative control.</p

Assessment of DNA quality obtained from each extraction method and impact on downstream applications.

<p>(A) Electrophoretic pattern of DNA extracts 1, 3, 4 and 5 of each extraction method on a 1% agarose gel. Ladder indicates a 1 kb DNA ladder as molecular weight size marker (B) 201 bp, 404 bp and 614 bp amplified DNA fragments of the <i>GAPDH</i> gene for sample 1, 3, 4, 5, of each extraction method. + indicates a positive control and – a negative control. (C) 125–175 bp multiplex PCR product for sample 1, 3, 4 and 5 of each extraction method. + indicates a positive control and – a negative control.</p

Sequences and product sizes of PCR and qPCR primers.

<p>F: Forward.</p><p>R: Reverse.</p

MET and HGF expressions.

<p>Representative sarcomas showing range and pattern of immunohistochemical staining for MET (A-H) and HGF (I-P). (A, B, I, J) staining intensity score = 0, (C, D, K, L) score = 1+, (E, F, G, M, N, O,) score = 2+, (H, P) score = 3+. (A, C, E, N) undifferentiated pleomorphic sarcomas, (B, D, F, G, K, M) angiosarcomas, (H) control cell line, (I, L, O) leiomyosarcomas, (J) dedifferentiated liposarcoma, (P) control tissue breast cancer.</p