Correlation between HOXA7 and HOXA9 (A) and JAG2 and HLF (B) gene expression in human AML analyzed with TaqMan Low Density Array (TLDA).

<p>Spearman rank correlation analysis was done on the log ratio values obtained from the TLDA assay and calculated with the 2^−ΔΔCT method (n = 34).</p

Venn diagram of genes significantly activated (A) or repressed (B) in Sca1+, Lin- BM cells expressing the ND13, NA10 or ND13(N51S) mutant fusion genes compared to GFP control.

<p>Venn diagram of genes significantly activated (A) or repressed (B) in Sca1+, Lin- BM cells expressing the ND13, NA10 or ND13(N51S) mutant fusion genes compared to GFP control.</p

Genes changed by NA10 and ND13 but not by the ND13(N51S) mutant compared to the GFP control.

<p>Genes changed by NA10 and ND13 but not by the ND13(N51S) mutant compared to the GFP control.</p

Gene expression levels in human AML relative to normal bone marrow.

<p>Gene expression levels in human AML relative to normal bone marrow.</p

Validation of microarray results with quantitative RT-PCR on unamplified RNA.

<p>Fold changes are calculated against an empty MIG control and <i>Gapdh</i> as endogenous control gene.</p

Hierarchical clustering of DNA methylation profiles.

<p>A) Unsupervised Euclidean hierarchical clustering was performed using beta values for the 1347 most variable CpG sites in T-ALL (standard deviation ≥0.30). In the dendogram each individual is represented as a vertical row and the specific CpG sites are shown in the horizontal columns. High methylation levels are shown in red and low levels in green, according to the Beta value scale bar in the figure where 1.0 is fully methylated and 0 is unmethylated. The T-ALL methylation subtypes are marked in the figure as low, intermediate and high, and with the merged classification in CIMP− and CIMP+ cases. The methylation status of the 1347 CpG sites of normal bone marrow samples (N1–N3) and two stimulated primary T-cell lines (P7/R2 and L2) are shown next to the hierarchical cluster. B−D) The mean CpG methylation levels within and outside CpG islands were examined. The distribution of mean methylation levels in low (n = 22), intermediate (n = 6) and high (n = 15) methylation subtypes are shown for: B) the entire array (26436 sites), C) within CpG islands (19211 sites), D) outside CpG islands (7225 sites). The significance (independent samples t-test) for differences in methylation between groups is shown.</p

Differently methylated genes in T-ALL are overrepresented by transcription factors and ligands and enriched for polycomb target genes.

<p>A) Lists of polycomb target genes identified in human embryonic fibroblasts and embryonic stem cells were compared with the DMGs in T-ALL in a Venn diagram. 260 genes were commonly present in these lists. B) The most variable CpG sites in T-ALL (1347 CpGs/1038 genes) and the 260 common polycomb genes identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065373#pone-0065373-g004" target="_blank">Figure 4A</a> were evaluated for protein function, and compared with the protein function distribution of genes within the GeneGO database (23868 genes) and the Illumina methylation array (15077 genes). Transcription factors and ligands were overrepresented in the DMG in T-ALL compared with the distribution within the database and the entire methylation array.</p

Clinical characteristics and outcome in 75 T-ALL of which 43 cases were CIMP classified.

<p>Clinical characteristics and outcome in 75 T-ALL of which 43 cases were CIMP classified.</p

Schematic overview of the methylation array analysis.

<p>Schematic overview of the methylation array analysis.</p

Event free survival (EFS) and overall survival (OS) in methylation subgroups in T-ALL.

<p>The 5 year EFS (A) and OS (B) for 43 T-ALL samples analyzed for CpG methylation and 32 T-ALL patients not analyzed but diagnosed during the same time-period showed no differences (p = 0.9 and p = 0.9 respectively). The total patient group (n = 75) had an EFS 5y of 0.59 (SE = 0.06) and OS 5y of 0.63 (SE = 0.06). CIMP+ (n = 21) and CIMP− (n = 22) patients differed significant regarding both 5 year EFS (C) and OS (D) (p = 0.001 and p = 0.02, respectively), with cases lacking methylation data forming an intermediate group.</p