Comparative analysis of CRY1 expression in a panel of different lymphoid malignancies.

<p>qRT-PCR analyses of PBMC samples obtained from patients with T-prolymphocytic leukemia (T-PLL), mantle cell lymphoma (MCL), plasma cell leukemia (PCL), hairy cell leukemia (HCL), B and T cell acute lymphoblastic leukemia (B-ALL, T-ALL), CLL and normal donors (ND), A. Red characters indicate samples that were further subjected to DNA methylation analysis of the CRY1 promoter, A. Analysis of CRY1 CpG island promoter methylation status, B. For experimental details and description of the graph in panel B refer to the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034347#pone-0034347-g002" target="_blank">Figure 2</a>.</p

Analysis of CRY1 CpG island promoter methylation status measured with the Bisulphite MassArray assay.

<p><b>A</b> Results of CRY1 CpG island methylation analysis performed on CLL samples and normal donors (ND) grouped by CD38 expression (CD38− samples, n = 28; CD38+ samples, n = 30; ND, n = 5). Each value represents the average amount of methylated CpGs of all analyzable CpGs within the CpG island promoter from one patient. The values represent the mean of duplicate experiments. The IgVH mutational status of each patient (if available) is highlighted in red; circles and squares indicate unmutated IgVH/V3-21 and mutated IgVH status, respectively. The median is marked as a line, error bars indicate SEM. Unpaired two-tailed t-test was used to compute p-values. <b>B</b> Samples from CLL patients and ND were subjected to both CRY1 mRNA expression and DNA methylation analysis with the bisulphite MassArray assay. mRNA expression values and percentage of methylated CpG were found to be highly correlated (r = −0.63, p<0.0001, Spearman correlation). The regression line in the plot was produced by linear regression analysis using promoter methylation as dependent and CRY1 mRNA expression as independent variable. <b>C</b> Correlation between the methylation data resulting from bisulphite genomic sequencing and the MassArray method showed high consistency (r = 0.86, p<0.0001, Spearman correlation).</p

Expression of CRY1 in CLL subgroups and normal donors.

<p><i>CRY1</i> mRNA expression in normal donors (ND, n = 35) in comparison to CLL samples from prognostic subgroups defined by CD38 expression (A, CD38+ samples, n = 36 vs. CD38− samples, n = 39) and IgVH mutational status (B, IgVH unmutated/V3-21, UM/V3-21, n = 23 vs. IgVH mutated, M, n = 18). mRNA levels are relative to GAPDH. Data are presented in a box-and-whisker format: the difference of the 25th and 75th percentile form the box (interquartile range, IQR), with the median marked as a line; the whiskers go down to the smallest value and up to the largest values. The Mann-Whitney U-test was used to compute p-values for pairwise comparisons.</p

Treatment-free survival according to the methylation status of the CRY1 promoter region.

<p>Treatment-free survival of 53 CLL patients with lowly methylated (n = 26) and highly methylated (n = 27) CRY1 promoter.</p

Patient characteristics.

*<p>n.a., not available.</p