Wnt pathway mutations do not contribute to the extent of dysregulated gene expression of Wnt pathway. (A) Expression profiles (from Affymetrix U133Plus2 arrays) of 60 Wnt pathway members that are significantly differentially expressed in 179 CLL-B cells compared with 24 normal CD19+ B cells (according to FDR corrected permutation test P values assessing significance of Student t test scores, FDR ≤0.05, with Student t test score ranging from −13.6-30.9). All tumor samples were comprised of >95% tumor purity. Genes were visualized in GENE-E. Wnt pathway genes downregulated in CLL are shown in the heatmap (top); Wnt pathway genes upregulated in CLL are shown (bottom). From these 60 genes, a “Wnt score” was calculated as a statistical measure of differential expression between the 37 known Wnt activators in the set (labeled in orange, in the column [right] of the heatmap) vs the 23 known repressors (labeled in black, separate column [right] of the heatmap). We generated a Student t test score for each CLL sample by comparing the differential expression of activators that are upregulated to repressors that are downregulated in each sample according to FDR-corrected permutation test P values assessing significance of Student t test scores, with FDR ≤0.05. Wnt scores ranged from −2.62 (blue) to 2.91 (red). Compared with normal B cells, overall, CLL cells demonstrate downregulation of Wnt pathway repressors and upregulation of Wnt pathway activators. (Bottom) CLL sample characteristics and whether samples also underwent whole-exome (WES) or whole-genome sequencing (WGS) (black-positive; white-negative). The rank of genes represented within the top 9% of genes differentially expressed array-wide is noted (in parentheses next to the gene names). Expression levels are log2 transformed and mean-centered for each gene for visualization. Induced levels are represented in red, repressed levels in blue, and no change is represented in white, in which levels are saturated at −0.5 and +0.5. (B) Unsupervised hierarchical clustering of Wnt pathway gene expression profiles from 12 mutated (with arrow) and 58 unmutated CLL-B cells (without arrow), performed using Pearson linear correlation with average linkage. (Right) Activators and repressors are shown (orange and black, respectively). (Upper panel) Wnt pathway genes and (lower panel) Wnt target genes, curated from the literature. A supervised analysis between the Wnt pathway mutated vs wild-type samples is presented in supplemental Figure 4. (C) The Wnt signaling pathway is chronically active in CLL-B cells. Normal and CLL-B cells were co-transfected with SuperTOPflash and pRL-TK constructs to measure endogenous TCF/LEF activity. CLL-B cells have fivefold greater normalized luciferase activity compared with normal B cells (n = 5; P < .01, Wilcoxon test, 2-tailed).