Gene expression profiling of marker genes in differentiated and undifferentiated MCT samples.

<p>Heat map and hierarchical clustering of all samples analyzed by DNA microarray using probes referable to the transcripts identified by class prediction analysis. Red indicates up-regulated and green down-regulated genes relative to the mean expression in all samples. For display purposes, samples in each class (differentiated and undifferentiated) were clustered separately and arranged from differentiated (left, yellow) to undifferentiated (right, red). Genes were hierarchically clustered separately based on the Pearson correlation coefficients and average linkage clustering. Units of the bar legend: absolute values.</p

Hierarchical clustering and heat map of differentiated and undifferentiated reference samples.

<p>Hierarchical clustering was performed using gene expression data of 597 differentially expressed genes obtained through the comparison of reference samples (13 differentiated and 5 undifferentiated mast cell tumors) with SAM, fixing a fold change of 2 as well as a False Discovery Rate of 0.01 as parameters. Red and green indicates up- and down-regulated genes relative to the mean expression in all samples, respectively. Samples were hierarchically clustered into differentiated (left, yellow) and undifferentiated (right, red) and based on the Pearson correlation coefficients and average linkage clustering. Genes were hierarchically clustered based on Pearson correlation coefficients and average linkage clustering. Units of the bar legend: absolute values.</p

Survival curves for mortality due to mast cell tumor, stratified by marker gene mRNA expression cut point.

<p>(A) FOXM1. The cut-off chosen was 0.5540 AU (FOXM1<0.5540 AU: n = 28; FOXM1>0.5540 AU: n = 23). (B) GSN. The cut-off chosen was 3.749 AU (GSN >3.749: n = 31; GSN <3.749 AU: n = 20). (C) FEN1. The cut-off chosen was 0.8155 AU (FEN1<0.8155 AU: n = 33; FEN1>0.8155 AU: n = 18). (D) KPNA2. The cut-off chosen was 0.4823 AU (KPNA2<0.4823 AU: n = 31; KPNA2>0.4823 AU: n = 20).</p

DNA microarray validation: Spearman correlation analysis of normalized DNA microarray data and corresponding qPCR results for the whole set of samples.

<p>***<i>p</i><0.001; statistical analysis was performed for each target gene-specific probe available on the array.</p

Target genes mRNA expression in differentiated and undifferentiated MCT samples.

<p>Data are expressed in arbitrary units. FC: -fold change.</p

Principal component analysis of differentiated and undifferentiated reference samples.

<p>Analysis was performed using gene expression data of 597 differentially expressed genes obtained through the comparison of reference samples (13 differentiated and 5 undifferentiated mast cell tumors) with SAM, fixing a fold change of 2 as well as a False Discovery Rate of 0.01 as parameters. Each colored sphere corresponds to a reference sample (differentiated mast cell tumors are indicated in yellow, while undifferentiated ones in red). The value of each principal component is reported on the graph. The sum of the three principal components accounted to the 79.883% of the total variance.</p

qPCR confirmatory analysis.

<p>Marker genes identified by class prediction analysis were amplified in an independent cohort of 22 mast cell tumors in order to comprehend their utility for mast cell tumor classification. Clustering analysis and PCA of gene expression data were performed using MultiD-Genex software for qPCR data, using the following settings: mean center scaling, Ward’s algorithm and Manhattan distance. (A) Clustering tree and (B) principal component analysis of independent mast cell tumor samples. Cases characterized by MCT-related death are indicated in red. The two groups identified are named group 1 and 2 (differentiated and undifferentiated MCTs, respectively). (C) Kaplan-Meyer survival plot stratified by molecular classification (group 1 and 2).</p

DAVID functional annotation of the complete list of differentially regulated genes between differentiated and undifferentiated MCTs.

<p>GO, Gene Ontology; BP: Biological Process; <i>p</i> value: modified Fisher exact P value calculated by DAVID software;</p><p>Fold Enrichment defined as the ratio of the two proportions: input genes involved in a biological process and the background information.</p

Survival curve of differentiated vs. undifferentiated mast cell tumors.

<p>Kaplan-Meyer survival plot stratified by molecular classification (differentiated and undifferentiated MCTs).</p