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

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

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

Progranulin Is a Novel Independent Predictor of Disease Progression and Overall Survival in Chronic Lymphocytic Leukemia

<div><p>Progranulin (Pgrn) is a 88 kDa secreted protein with pleiotropic functions including regulation of cell cycle progression, cell motility, wound repair and tumorigenesis. Using microarray based gene expression profiling we have recently demonstrated that the gene for Pgrn, granulin (<i>GRN)</i>, is significantly higher expressed in aggressive CD38⁺ZAP-70⁺ as compared to indolent CD38⁻ZAP-70⁻ chronic lymphocytic leukemia (CLL) cases. Here, we measured Pgrn plasma concentrations by enzyme-linked immunosorbent assay (ELISA) in the Essen CLL cohort of 131 patients and examined Pgrn for association with established prognostic markers and clinical outcome. We found that high Pgrn plasma levels were strongly associated with adverse risk factors including unmutated IGHV status, expression of CD38 and ZAP-70, poor risk cytogenetics (11q-, 17p-) as detected by flourescence in situ hybridization (FISH) and high Binet stage. Pgrn as well as the aforementioned risk factors were prognostic for time to first treatment and overall survival in this series. Importantly, these results could be confirmed in the independent multicentric CLL1 cohort of untreated Binet stage A patients (n = 163). Here, multivariate analysis of time to first treatment revealed that high risk Pgrn (HR = 2.06, 95%-CI = 1.13–3.76, p = 0.018), unmutated IGHV status (HR = 5.63, 95%-CI = 3.05–10.38, p<0.001), high risk as defined by the study protocol (HR = 2.06, 95%-CI = 1.09–3.89, p = 0.026) but not poor risk cytogenetics were independent prognostic markers. In summary our results suggest that Pgrn is a novel, robust and independent prognostic marker in CLL that can be easily measured by ELISA.</p></div

Association of Pgrn plasma levels and clinical outcome in the CLL cohort from Essen.

<p>Kaplan-Meier curves depict the cumulative proportion of untreated (TTFT, A) and surviving (OS, B) patients with CLL. Statistical comparisons between patients with high (>165.5 ng/ml) and low Pgrn plasma levels (≤165.5 ng/ml) were performed using the log-rank test.</p

Association of Pgrn plasma levels and clinical outcome in the multicentric CLL1 cohort.

<p>Kaplan-Meier curves depict the cumulative proportion of progression free survival (PFS, A), time to first treatment (TTFT, B) and overall survival (OS, C) of patients with CLL. Statistical comparisons between patients with high (>79.2 ng/ml) and low Pgrn plasma levels (≤79.2 ng/ml) were performed using the log-rank test.</p

Patient characteristics of the CLL cohort from Essen.

*<p>Leukemic samples exhibiting a 17p- and/or 11q- karyotype were assigned to the high risk and samples with either no chromosomal abnormalities or aberrations of chromosomes 13q and 12 to the low risk category. Leukocyte counts and age were assessed at the time of sample acquisition whereas all other risk parameters were determined at the time of diagnosis.</p

Correlation of Progranulin plasma levels and established prognostic markers in the CLL cohort from Essen.

<p>Plasma samples collected from high-risk patients defined by the presence of unmutated IGHV genes (A), presence of del17p and/or del11q as detected by iFISH (B), expression of CD38 (C) and ZAP-70 (D), low percentage of smudge cells (E) as well as advanced Binet stage B/C (F) exhibited significantly higher Pgrn plasma concentrations as compared to low-risk patients. Statistical comparisons were made using the Mann-Whitney U-test (A–E) and Kruskall Wallis test (F). The bold horizontal bars represent the median and the whiskers indicate the interquartile range.</p