ABSTRACT: Global gene expression analysis was performed in cases 1, 2, 5, 6 and 8 as well as in five pooled myxofibrosarcomas using the Affymetrix GeneChip Human Gene 1.0 ST Arrays (Affymetrix Inc., Santa Clara, CA, USA). Unsupervised hierarchical clustering analysis, using Pearson correlation and average linkage clustering, separated the control pool and case 6 from the rest of the cases. Cases 1 and 5, both harboring t(1;10)(p22;q24), were in turn segregated from cases 2 and 8. Tumors with t(1;10)(p22;q24) thus seemed to have a more distinct expression pattern than tumors affected by amplification of regions on chromosome 3. Tumors with t(1;10) showed increased expression levels of NPM3 and FGF8 compared with tumors without such a translocation, including the control pool. No other gene in the 5 Mb region proximal to the breakpoint on chromosome 10 displayed a more pronounced difference between the two groups. Of the genes located in the commonly amplified region on chromosome 3, VGLL3 and CHMP2B showed a higher expression in tumors affected by the amplification. Cases 1, 2, 5, 6 and 8 were analyzed using the GeneChip Human Gene 1.0 ST Arrays (Affymetrix Inc, Santa Clara, CA, USA). As a control, a pool of five myxofibrosarcomas was used. Total RNA was extracted from frozen tumor biopsies using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and RNeasy (Qiagen, Valencia, CA, USA) according to the manufacturers’ instructions. RNA quality and concentration were measured using an Agilent 2100 bioanalyzer and Nanodrop ND-1000, respectively. cDNA was generated with the GeneChip® Whole Transcript (WT) cDNA Synthesis and Amplification Kit (Affymetrix) using 300 ng total RNA. Amplified cDNA was fragmented and end labeled using the GeneChip® WT Terminal Labelling Kit (Affymetrix). Subsequently, the fragmented and biotinylated cDNA was hybridized to the GeneChip® Human Gene 1.0 ST Arrays (Affymetrix). The arrays were washed and stained on a GeneChip® Fluidics Station 450 (Affymetrix) according to the manufacturer’s recommendations. Scanning was carried out with the GeneChip® Scanner 3000 and image analysis was performed using GeneChip® Operating Software (GCOS, Affymetrix). Expression data was normalized, background corrected and summarized using the RMA algorithm implemented in the Affymetrix Expression Console™ version 1.0 software. Clustering was performed with MeV 4.1.01 software (http://www.tm4.org/mev.html), using unsupervised hierarchical clustering analysis on the basis of Pearson correlation and average linkage clustering.
