BackgroundFludarabine, is one of the most active single agents in the treatment of chronic lymphocytic leukemia (CLL). Over time, however, virtually all CLL patients become fludarabine-refractory. To elucidate whether microRNAs are involved in the development of fludarabine resistance, we analyzed the expression of 723 human miRNAs before and 5-days after fludarabine mono-therapy in 17 CLL patients which were classified as responder or refractory to fludarabine treatment based on NCI criteria.ResultsBy comparing the expression profiles of these two groups of patients, we identified a microRNA signature able to distinguish refractory from sensitive CLLs. The expression of some microRNAs was also able to predict fludarabine resistance of 12 independent CLL patients. Among the identified microRNAs, miR-148a, miR-222 and miR-21 exhibited a significantly higher expression in non-responder patients either before and after fludarabine treatment. After performing messenger RNA expression profile of the same patients, the activation of p53-responsive genes was detected in fludarabine responsive cases only, therefore suggesting a possible mechanism linked to microRNA deregulation in non-responder patients. Importantly, inhibition of miR-21 and miR-222 by anti-miRNA oligonucleotides induced a significant increase in caspase activity in fludarabine-treated p53-mutant MEG-01 cells, suggesting that miR-21 and miR-222 up-regulation may be involved in the establishment of fludarabine resistance.ConclusionsThis is the first report that reveals the existence of a microRNA profile that differentiate refractory and sensitive CLLs, either before and after fludarabine mono-therapy. A p53 dysfunctional pathway emerged in refractory CLLs and could contribute in explaining the observed miRNA profile. Moreover, this work indicates that specific microRNAs can be used to predict fludarabine resistance and may potentially be used as therapeutic targets, therefore establishing an important starting point for future studies.

Cytogenetic and fluorescence in situ hybridization studies were successfully performed in 217 chronic lymphocytic leukemia (CLL). In all, 13 patients with 6q21 deletion were identified and characterized in comparison with 92 patients with 'favourable' karyotype (normal or 13qÀ), 69 cases with 'intermediate risk'(1-2 anomalies) and 43 cases with 'unfavourable' karyotype (complex, 11qÀ or 17pÀ). Six out of 13 cases with 6qÀ showed an excess of atypical lymphocytes, a finding confirmed at the histologic level; 420% CD38 þ cells were seen in 5/6 cases. IGVH mutational status revealed 498% homology to the germline sequence in 4/10 cases. When compared with the 'favourable' group, patients with 6qÀ showed a higher white blood cell (WBC) count, frequent splenomegaly, atypical morphology, CD38 þ and short time from diagnosis to first treatment and short survival. A higher median WBC count was found in the 6qÀ group vs the intermediate-risk group; survival was shorter in the unfavourable group. To ascertain if the 6qÀ anomaly was an independent factor predicting for an inferior outcome among those patients with 'favourable' cytogenetics, we performed an analysis of prognostic factors in 105 patients (92 'favourable' plus 13 with 6qÀ), showing that the 6qÀ chromosome maintained its prognostic significance at multivariate analysis (P ¼ 0.02) along with stage (P ¼ 0.01). We conclude that CLL with 6qÀ is characterized by a high incidence of atypical morphology, classical immunophenotype with CD38 positivity and intermediate incidence of IGVH somatic hypermutation. Clinicobiological features and outcome show that this cytogenetic subset of CLL should be allocated in an intermediate-risk category.

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It is unclear whether karyotype aberrations that occur in regions uncovered by the standard fluorescence in situ hybridization (FISH) panel have prognostic relevance in chronic lymphocytic leukemia (CLL). We evaluated the significance of karyotypic aberrations in a learning cohort (LC; n ‫؍‬ 64) and a validation cohort (VC; n ‫؍‬ 84) of patients with chronic lymphocytic leukemia with "normal" FISH.