Pathway signature of VEGF and NOTCH mediated EMT in ccRCC.

<p>Comparison of gene expression data from the FFPE and from the RNAlater^® dataset with published results [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149743#pone.0149743.ref020" target="_blank">20</a>] and between themselves. <i>F = FFPE samples</i>, <i>R = RNAlater</i>^<i>®</i> <i>samples</i>, <i>Numbers = fold change of up-regulation (red) or down-regulation (blue)</i>.</p

Immunohistochemistry and mRNA plots.

<p>(A) Immunohistochemistry of UMOD, NTPX2 and CA9. <i>Magnification x20</i>, <i>scale bar 50 μm</i>. (B) Respective mRNA abundance plots in the FFPE and in the RNAlater^® datasets.</p

Gene network.

<p>The most differentially affected network with the central role of <i>TGFB1</i> in (A) FFPE samples and B) RNAlater data sets. <i>Proteins with cancer involvement are marked with purple outline</i>. <i>Red fill indicates overrepresentation of the gene in ccRCC</i>, <i>green indicates under-representation</i>. <i>Color intensity reflects range of fold change</i>.</p

Characteristic patient features at the time of surgery.

<p>eGFR was calculated with the MDRD formula. The staging was performed based on the EAU Guidelines on renal cell carcinoma: 2014 update [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149743#pone.0149743.ref043" target="_blank">43</a>].</p

Correlation of gene expression data.

<p>The correlation of commonly differentially expressed genes is given with respect to (A) average expression and (B) log2 fold changes.</p

Gene expression analyses.

<p>The 20 most up- or down-regulated genes in the FFPE data set with corresponding RNAlater^® values (upper panel), and the 20 most up- or down regulated genes in the RNAlater^® dataset with corresponding FFPE values (lower panel), filtered by adjusted p-value≤0.05. Rank indicates the rank of the gene within the list of differentially genes sorted by largest to smallest absolute fold change. 14 genes are shared between the two lists. <i>TU</i>: <i>tumour</i>, <i>NO</i>: <i>normal</i>, <i>FC</i>: <i>fold change</i>, <i>ND</i>: <i>not detected</i>, <i>did not pass the expression filter</i>.</p

Development of a candidate marker for ccRCC.

<p>(A) Expression values of <i>CA9</i> correctly classified 30 of 32 samples in our FFPE dataset. (B) Whisker plot of expression value distribution in our FFPE dataset for <i>CA9</i>. (C) Scatterplot for the expression values of <i>CA9</i> in our FFPE and in our RNAlater dataset. (D) <i>CA9</i> expression values correctly classify 139 out of 144 samples in a microarray dataset of ccRCC (GSE53757). (E) Distribution of <i>CA9</i> expression values for normal (NO) and ccRCC tumor samples (TU) in the GSE53757 dataset. (F) Stratification of the expression values of overexpressed <i>CA9</i> into all four stages of ccRCC [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149743#pone.0149743.ref014" target="_blank">14</a>].</p

Comparison of our gene expression data with data from literature [17].

<p>Twenty genes with smallest p-values and largest absolute fold changes in a meta-analysis of five microarray studies are compared to the corresponding genes and their fold changes and p-values of the NGS datasets. The median fold changes and standard deviations for the meta-analysis are presented. All shown genes were differentially expressed in only 2 or 3 microarray datasets. Large standard deviations indicate a large spread of values in the individual microarray studies. 17 of the 20 genes were found differentially expressed in both NGS datasets, 13 of these with fold changes within the fold change range of the microarray meta-analysis. <i>ND</i>: <i>not detected</i>, <i>did not pass initial expression filter</i>.</p

Multidimensional scaling (MDS) analysis of gene expression data.

<p>MDS analysis based on all commonly detected genes shows that samples segregate by diagnosis (A) and not by storage condition (B). Distances correspond to leading log-fold-changes between each pair of samples. MDS based on differentially expressed genes demonstrates less within-group variance compared to MDS with all detected genes in the RNAlater^® (C) and FFPE (D) datasets. <i>NF</i>: <i>Normal</i>, <i>FFPE; NR</i>: <i>Normal</i>, <i>RNAlater</i>^<i>®</i><i>; TF</i>: <i>Tumor</i>, <i>FFPE; TR</i>: <i>Tumor</i>, <i>RNAlater</i>^<i>®</i>. <i>NO = Normal; TU = Tumor</i>.</p

Pathway analysis.

<p>The 20 most affected canonical pathways in each NGS dataset with the corresponding values and ranks. Rank indicates the place of the pathway within the list of pathways sorted by largest to smallest –log(adjusted p-value). 12 of 20 pathways are shared between both datasets. <i>TU</i>: <i>tumour</i>, <i>NO</i>: <i>normal</i>, <i>FC</i>: <i>fold change</i>, <i>ND</i>: <i>not detected</i>, <i>did not pass the expression filter</i>.</p