MicroRNA in Chronic Lymphocytic Leukemia Figure 1. GSEA reveals a B cell activation miRNA signature in CLL. A. Heatmap of B cell activation signature miRNAs comparing expression in unstimulated control B (CB) cells and CpG activated B cells (CB act). B. GSEA enrichment profile of downregulated B cell activation signature miRNAs in control B cells versus CLL cells. C. Heatmap of downregulated B cell activation signature miRNA expression in control B cells and CLL cells. D. GSEA enrichment profile of upregulated B cell activation signature miRNAs in control B cells versus CLL cells. E. Heatmap of upregulated B cell activation signature miRNA expression in control B cells and CLL cells. doi:10.1371/journal.pone.0016956.g001 27b, miR-181a, miR-181b, and miR-223 (all downregulated in the CLL signature) and increased miR-155 (upregulated in the CLL signature), regardless of the mechanism of B cell activation (Figure S7 and summarized in Table 1). Most miRNAs demonstrated consistently altered expression regardless of the mechanism of B cell activation though the expression of the miR-29 family varied depending upon B cell activation. Whereas anti-IgM and CD40L upregulated miR-29a (Figure S7A and S7B), LPS, CpG, or polyI:C downregulated miR-29a (Figure S7C). In contrast, anti- IgM and CD40L downregulated miR-29b and miR-29c (Figure S7A and S7B) but LPS, CpG, or polyI:C upregulated miR-29b and miR-29c (Figure S7C). miR-223 is increased in response to CD40L activation (Figure S7B), while decreased in response to all other stimuli (Figure S7A and S7C). Additionally, the expression of miR-26a was increased in CLL and only in B cells activated with CD40L but did not change in B cells activated with anti-IgM (Figure S7A and S7B). These data suggest that particular CLL signature miRNAs are altered in response to specific B cell stimuli. In contrast, miR-150 was reduced during B cell activation, and upregulated in almost all the CLL samples tested (Table 1). Thus, high expression of miR-150 may be CLL specific. Signature miRNA alterations are associated with clinical features and time to first therapy Previous reports suggest that altered miRNA expression has prognostic value [30,31,32]. ZAP70 and IgVH mutational status are strongly associated with clinical outcome for CLL patients. Thus, comparative marker alignments were performed to identify miRNAs clustered by ZAP70 and IgVH status (Table S4A and S4B). Out of 38 patients, we examined 14 ZAP70+ patients and 14 IgVH unmutated patients (Table S1). We found that miR-150 was upregulated in ZAP70+ patients whereas miR-29c and miR-223 were upregulated in both ZAP702 and in IgVH mutated patients suggesting that decreased levels of miR-150 and increased levels of miR-29c and miR-223 may be associated with better clinical outcomes. Our data do not indicate a statistically-significant correlation between miR-29c and miR-223 with any established cytogenetic abnormalities used to distinguish CLL patient sub- populations, such as 17p, 11q and 13q deletion and trisomy 12 (data not shown). Additionally, miR-92a was upregulated in ZAP702 patients and let-7g was upregulated in IgVH mutated patients (Table S4A and S4B). We also examined miRNA expression across CLL cells for association with time to first therapy in these patients. Using recursive partitioning analysis for finding optimal cut points for miRNA expression, we examined time to first therapy. We identified two miRNAs (miR-29c and miR-223) with q-values of 0.07 associated with an optimized binary split (Kaplan-Meier curves and the optimal splits are indicated (Figure 4)). CLL patients with lower expression levels of these miRNAs demonstrated shorter time to first therapy compared to CLL patients who did not demonstrate this pattern of miRNA expression. Decreased expression of these PLoS ONE | www.plosone.org 4 March 2011 | Volume 6 | Issue 3 | e16956