variables, and the Kruskal-Wallis test for variables with three or more categories. Time to first therapy was calculated as time from initial diagnosis to first therapy; patients not yet treated were censored at date last known alive. Time to first therapy was estimated using the method of Kaplan and Meier; the log rank test was used to assess associations with time to first therapy. Recursive partitioning was used to identify an optimal binary split for each miRNA individually, using the rpart package in R. P-values from recursive partitioning are not adjusted for the optimization of the method. Two-sided p-values are not adjusted to reflect multiple comparisons; q-values reflect adjustment for multiple comparisons using the false discovery rate of Benjamini and Hochberg, as implemented in the q-value package in R. A false discovery rate of 0.10 or smaller was the criterion for reporting significant differences in both clinical features and time to first therapy in this study. Results Determinants of a CLL-specific miRNA expression signature CLL cells are characterized by expression of both CD5 and CD19 cell surface markers [20]. CD19 bead positive selection was used to purify CLL cells and greater than 97% CLL cell purity was verified by FACS analysis of CD5 and CD19 expression for all samples used in this study (Figure S1). We profiled miRNA expression across 38 highly purified CLL patient samples, 9 control B cell samples and 5 activated B samples by CpG using the Luminex method [18]. Unsupervised hierarchical analysis clus- tered CLL samples based upon miRNA expression (Figure S2). Comparative marker selective view alignment analysis in CLL samples compared with control B samples identified a CLL- specific miRNA signature consisting of upregulation of let-7g, miR-26a, miR-29a, miR-29b, miR-29c, miR-101, miR-150, and miR-155, and downregulation of miR-23a, miR-24, and miR-27b, miR-181a, miR-181b, and miR-223 (Figure S3). Though most B cells are CD5-CD19+, specific subsets of B cells (e.g. peritoneal, tonsillar) are CD5+CD19+. To verify that miRNA expression profiling discerned differences between normal CD5- CD19+ B cells and CD5+CD19+CLL cells rather than between normal CD5-CD19+ and CD5+CD19+ B cells, control B cells from 4 different control donors were sorted into CD5- and CD5+ populations and RT-PCR analysis was performed. Our data indicate that miR-181a and miR-181b were downregulated in 6 CLL samples relative to both CD5- and CD5+ control B cell populations. Similarly, miR-29a, miR-150, and miR-155 were upregulated in CLL relative to both CD5- and CD5+ control B cell populations (Figure S4). These data indicate that CD5+ expression status does not affect the interpretation of the changes in miRNA expression in these studies. Notably, CD19 positive purification does not alter miRNA expression as assessed by RT-PCRs (Figure S5). miRNA alterations identify an activated B cell phenotype in CLL cells Several characteristic miRNAs that have altered expression in CLL relative to untransformed B cells are also important for lymphocyte activation (e.g. miR-181a [21], miR-181b [22], miR- 150 [23], and miR-155 [24]), inflammatory responses (e.g. miR- 155 [25,26]), and B lymphoproliferative disorders (eg. miR-155 [27,28]). We thus hypothesized that CLL cells may derive from partially activated B cells. To systematically compare miRNA expression in CLL to the miRNA changes induced by B cell activation, we identified sets of miRNAs significantly (p,0.05) upregulated or downregulated after MicroRNA in Chronic Lymphocytic Leukemia untransformed B cell activation by CpG. The upregulated miRNAs were miR-34a, miR-198, miR-155, miR-337-3p and miR-342-3p and the downregulated miRNAs were let-7c, miR- 15b, miR-20b, miR-103, miR-181a, miR-181b, and miR-331-3p (Figure 1A). We then performed GSEA for these upregulated and downregulated miRNAs in CLL versus untransformed B cells (Figure 1B and 1D). Four out of seven downregulated miRNAs (miR-15b, miR-103, miR-181a, and miR-181b) were expressed at lower levels in CLL, and five out of five upregulated miRNAs (miR-34a, miR-155, miR-198, miR-337-3p and miR-342-3p) were expressed at higher levels in CLL compared to untransformed B cells (Figure 1C and 1E). Next, we used miRNA-specific RT-PCR to confirm the expression of these signature miRNAs in 3 control B samples and 3 activated B samples (different from the samples used in the miRNA profiling) after CpG activation (Figure 2A, 2B, and 2C) and in control B samples and at least 4 CLL patient samples per miRNA, (Figure 2D and 2E). Using RT-PCR, we independently confirmed these miRNA expression patterns in control B and activated B cells (Figure 2A and 2B). However, in CLL cells, miR- 337-3p demonstrated an opposite trend to the one identified in GSEA of miRNA expression profiling (Figure 1E). Moreover, miR-15b and miR-198 demonstrated a similar trend in as in GSEA of miRNA expression profiling (Figure 1C and 1E) though the p value was not statistically significant (Figure 2D and 2E). Variations detected between Luminex bead-based profiling and RT-PCR may be due to the heterogeneity of CLL as we used a subset of CLL samples for the qPCR confirmation. To mechanistically link altered miRNA expression in CLL with altered expression of miRNAs observed in B cell activation, we carefully examined the expression of one miRNA (miR-155) whose expression is increased in CLL and in activated B cells. The miR- 155 gene is activated upon B cell stimulation and contains binding sites for the AP-1 transcription factor. B cell activation stimulates the JNK pathway, increases the levels of phospho-ERK, and then activates AP-1 [29]. Treatment of CpG activated B cells and CLL cells with either JNK or MEK inhibitor decreased the expression of miR-155 (Figure S6A and S6B). These data indicate common signaling pathways affect altered miRNA expression observed in activated B cells and CLL cells. To confirm the activation phenotype indicated by miRNA expression profiling, we performed FACS analysis of na¨ıve B cells, activated B cells, and CLL cells using B cell activation markers CD69, CD80, and CD86 (Figure 3 and Table S3). Our data demonstrate that CLL cells express CD69 and CD80 at levels that approximate the levels observed in activated B cells and that CLL cells express CD86 at levels intermediate between na¨ıve and stimulated B cells. These data indicate that CLL cells have similar gene expression patterns as activated B cells. To independently verify that miRNA changes observed in CLL cells are characteristic of an activated B cell status, we purified B cells from healthy donors and stimulated these cells with a variety of B cell activators including anti-IgM and CD40L (BCR and T cell- assisted co-stimulatory pathways), and LPS, CpG, or polyI:C (Toll- like receptor pathways) and examined miRNA expression. Stimulation of highly purified, control B cells was verified by FACS analysis of cell membrane expressed activation markers including CD69, CD80 and CD86 expression (Figure 3 and Table S3). In addition to the activation signature, additional miRNAs are differently expressed in CLL compared to untransformed B cells. We tested the expression patterns of these miRNAs to determine if they also were altered in untransformed B cells upon activation. For the CLL signature miRNAs, we found that activation of control B cells led to reduced miR-23a, miR-23b, miR-24, miR- PLoS ONE | www.plosone.org 3 March 2011 | Volume 6 | Issue 3 | e16956